ES2341578T3 - PROPHONGED LIBERATION MICROSPHERES. - Google Patents

Abstract

Microsphere comprising: (1) a protein or carrier molecule selected from the group consisting of IgG; IgM; insulin; hGH; lysozyme; alpha-lactoglobulin; basic fibroblast growth factor; VEGF; chymotrypsin; trypsin, carbonic anhydrase; ovalbumin; phosphorylase b; alkaline phosphatase; beta-galactosidase; fibrinogen; poly-L-lysine; DNA; immunoglobulins (eg, antibodies); casein; collagen; Soy protein; and jelly .; (2) a water soluble polymer; (3) a first complexing agent that is a polyanionic polysaccharide and (4) a second complexing agent that is a divalent metal cation selected from the group consisting of calcium and magnesium

Description

Prolonged release microspheres.

Field of the Invention

The invention relates to methods and compositions for forming and using release microspheres prolonged The microspheres are porous and include multiple corrugated holes with a diameter of less than 1,000 angstroms.

Background of the invention

The microparticles, microspheres and microcapsules, collectively referred to herein as "microparticles", they are solid or semi-solid particles that have a diameter less than one millimeter, preferably less than 100 microns, the which can be formed by various materials, including synthetic polymers, proteins and polysaccharides. The microparticles have been used in numerous applications different, mainly separations, diagnoses and supply of medications.

The best known examples of microparticles employed in separation techniques are those that are formed by polymers of both synthetic and protein origin, such as polyacrylamide, hydroxyapatite or agarose. These polymeric microparticles are normally used to separate molecules, such as proteins, according to molecular weight and / or ionic charge or by interaction with molecules chemically coupled to microparticles.
asses

In the field of diagnosis, the microparticles they are frequently used to immobilize an enzyme, the substrate for an enzyme or a labeled antibody, which is then subjected to the interaction with a molecule to be detected directly or indirectly.

In the area of controlled supply of medications, the molecules are encapsulated within the microparticles or are incorporated into a monolithic matrix for post release Several different ones are usually used techniques to make these microparticles from polymers synthetic, of natural polymers, of proteins and polysaccharides, including phase separation, solvent evaporation, an emulsification and spray drying. Generally the polymers form the support structure of these microspheres and the medicine of interest is incorporated into the polymer structure. Examples of polymers used for microsphere formation include homopolymers and copolymers of lactic acid and acid glycolic (PLGA), as described in the US Pat. United States No. 5,213,812 to Ruiz, in United States Patent No. 5,417,986 to Reid et al., In U.S. Patent No. 4,530,840 to Tice et al., U.S. Patent No. 4,897,268 of Tice et al., U.S. Patent No. 5,075,109 to Tice and col., U.S. Patent No. 5,102,872 to Singh et al., U.S. Patent No. 5,384,133 to Boyes et al., Patent of U.S. No. 5,360,610 to Tice et al., and in the Publication of European Patent Application Number 248,531 of Southern Research Institute; block copolymers such as tetronic 908 and poloxamer 407, as described in US Patent No. 4,904,479 of Illum; and polyphosphazenes, as described in the Patent of United States No. 5,149,543 to Cohen et al. Microspheres made by using polymers such as these they have little efficiency in front of the load and, often, they are only capable of incorporating a small percentage of the drug of interest in the polymeric structure. Therefore, they must often be administered substantial amounts of microspheres to achieve an effect therapeutic.

The beads or spherical particles have been commercially available for many years as a tool for biochemists For example, antibodies conjugated with beads originate relatively large particles specific to ligands private individuals Normally large coated particles are used of antibodies to cross-link the receptors on the surface of a cell for cell activation, they are linked to a phase solid for immunoaffinity purification and can be used to deliver a therapeutic agent that is slowly released into time, using specific tissue or tumor antibodies conjugated with the particles to direct the agent towards the site wanted.

The most common method of covalent bonding of a antibody to a solid phase matrix consists of derivatizing a pearl with a chemical conjugation agent and then bond the antibody to activated pearl. The use of a pearl polymeric synthetic and not of a protein molecule allows to use derivatization conditions much more severe than those that may withstand many proteins, is relatively cheap and often generates  a link that is stable in a wide spectrum of conditions denaturing Various pearls are commercially available derivatized, all with different constituents and sizes. The beads formed from synthetic polymers, such as polyacrylamide, polyacrylate, polystyrene or latex, are commercially available from numerous sources, such as Bio-Rad Laboratories (Richmond, Calif.) And LKB Produkter (Stockholm, Sweden). Pearls formed from macromolecules and natural particles such as agarose, agarose cross-linked, globulin, deoxyribonucleic acid and liposomes are commercially available from sources such as Bio-Rad Laboratories, Pharmacia (Piscataway, N.J.) and IBF (France). The beads formed from polyacrylamide and agarose copolymers They are commercially available from sources such as IBF and Pharmacia. Magnetic beads are commercially available from sources as Dynal Inc. (Great Neck, N.Y.).

A disadvantage of microparticles or beads currently available is that they are difficult and expensive to produce. The microparticles produced by means of these methods known have a wide distribution of particle sizes, to they often lack uniformity and fail to present a kinetic of long-term release when the concentration of Active ingredients is high. In addition, the polymers used in these known methods are dissolved in organic solvents with the In order to form the microspheres. Therefore, the microspheres must be developed in special facilities designed to handle organic solvents These organic solvents could denature the proteins or peptides contained in the microparticles The residual organic solvents could be toxic when administered to humans or animals.

In addition, the microparticles available rarely they are small enough for just pierce the holes the size of a needle commonly used to administer therapeutics or as to be useful in administration via inhalation. For example, the microparticles prepared using polylactic glycolic acid (PLGA) are large and tend to aggregate. A stage of size selection, resulting in a loss of product, to remove those particles too large for your injection. Those PLGA particles with a size suitable for your injection should be administered using a large needle caliber to correspond to the large particle size, with often causing discomfort to the patient.

Generally, all microspheres currently available are activated to release your media content aqueous and, therefore, should be lyophilized to prevent their premature release Also, particles like those prepared using the PLGA system have a kinetics of release based on both erosion and diffusion. In this type of system, an initial explosion or release is observed Quick medication. This explosion effect may result in unwanted side effects in those patients who are They have administered the particles.

Currently, microparticles are prepared using lipids to encapsulate target medications. For example, to encapsulate water soluble drugs for the purpose of their subsequent supply, as described in US Patent No. 5,422,120 to Sinil Kim. In general, these particles are larger than 10 µm in size and are designed for joint, intrathecal, subcutaneous or epidural administration. As an alternative, liposomes have been used for intravenous delivery of small molecules. Liposomes are spherical particles composed of multiple or simple bilayers of phospholipids and cholesterol. Liposomes are 30 µm in size or larger and can carry a variety of water-soluble or lipid-like medications. Liposome technology has been hampered by problems that include the purity of lipid components, possible toxicity, heterogeneity and stability of the vesicles, excessive absorption and difficulties with their manufacture or life.
Useful.

Therefore, there is a growing need for develop new methods of manufacturing microparticles, in particular that can be adapted to its use in separations, in the diagnostic and drug supply field. Preferably, these improved microparticles would allow prolonged release of active agents in a way Predictable and consistent.

Summary of the invention

The invention solves these and other problems by providing methods and compositions for the prolonged release of therapeutic and / or diagnostic agents in vivo and in vitro . As used hereinafter, the term "therapeutic agent" is intended to include clinical agents that can be administered in microcapsular form when used essentially for treatment or diagnosis.

According to one aspect of the invention, provides a smooth surface microsphere that includes multiple  corrugated holes. The corrugated holes have a diameter less than 1,000 angstroms, as determined by the gas adsorption technique to determine pore size. The Microspheres of the invention include a macromolecule, preferably a protein or a nucleic acid, and at least one water soluble polymer. In general, microspheres form contacting the macromolecule and at least one polymer soluble in water under aqueous conditions to form the microspheres, and then the microspheres are stabilized both by chemical crosslinking as per its exposure to an energy source, preferably of heat, or both, at a certain temperature, resulting in microspheres that are resistant to treatments physical and chemical such as sonication and caustic solutions Under these conditions. Although you do not want a link to no particular theory or mechanism, it is thought that microspheres are formed during the mixing stage; but nevertheless, these initially formed microspheres are transient and require another stage (for example, include an agent of cross-linking in the mixture and / or applying heat or other source of energy) to stabilize such microspheres temporarily formed. The particular conditions to form the microspheres Representative of the invention are described in the Examples. In the preferred embodiments, the formation reaction is carried out no added oils or organic solvents. Oil is defined as a substance that is liquid fat, which is insoluble in Water.

The protein component of the microsphere can be a carrier protein or a therapeutic protein (see, for example, Table 1). As used herein, a "carrier protein" refers to a protein that has a molecular weight of approximately at least 1,500 and is presented in three-dimensional structure. The carrier protein may also be a therapeutic protein, that is, a protein that has therapeutic activity; however, in general, the term "carrier protein" will be used in this application to refer to a protein that has an essential function to provide a three-dimensional structure for the purpose of microsphere formation, even though the carrier protein may also have a secondary function as a therapeutic agent. In certain preferred embodiments, the carrier protein is an albumin, in particular human serum albumin. Optionally, the protein microspheres of the invention further include a therapeutic agent such as a steroid (eg estradiol, testosterone, prednisolone, dexamethasone, hydrocortisone, lidocaine base, procaine base) or any other chemical entity known to bind to the protein, preferably albumin, such as GCSF or paclitaxel. In still other embodiments (set forth below), the microspheres of the invention further include a complexing agent and, preferably, a therapeutic agent (preferably a peptide) that is associated with the complexing agent by an ionic or non-ionic interaction. In certain embodiments, the protein comprising the matrix is a therapeutic protein (for example a hormone such as insulin or human growth hormone) and the microsphere is constructed and arranged to provide a prolonged release of the therapeutic protein in vivo . Preferably, the microsphere is constructed and arranged to provide a prolonged release of the therapeutic agent in the absence of a significant increase in the microsphere's volume.

Surprisingly, the surface of the Microsphere is different from its interior. A strong wash with water of the microspheres broken by freezing dissolves much of the material of the microsphere matrix, leaving a wrap fine. In addition, the surface of the microsphere is smooth; the holes Corrugated (pores) have a diameter of less than 1,000 angstroms, as determined by gas adsorption techniques for pore size determination, using for example the BET technology for data analysis.

In general, the microspheres of the invention will be formed by mixing the macromolecule, preferably a protein or a nucleic acid, with at least one low aqueous polymer conditions that allow the water soluble polymer to eliminate water ("dehydrate") of the macromolecule, within specific or preferred proportions between the macromolecule and the water soluble polymer. As used herein, a "polymer water soluble "of the invention refers to a polymer or to a mixture of polymers that are capable of removing water or dehydrate the macromolecule or that is otherwise capable of cause a volume exclusion. Thus, the preferred process involves the exclusion of volume by using a fully aqueous system without involving any oil or solvent organic.

Suitable water soluble polymers include soluble branched or linear polymers, preferably those of high molecular weight. The polymers can be very water soluble, moderately soluble in water or slightly water soluble (water solubility greater than 2% weight / volume) Preferred water soluble polymers are water soluble or soluble in a water miscible solvent. The Water soluble polymers can be solubilized first by their dissolution in a water miscible solvent and then combining the polymer solution with an aqueous solvent. In the particularly preferred embodiments, soluble polymers in water of the invention are selected from among the polymers water soluble shown in Table 2. In certain embodiments, the microspheres of the invention are formed by proteins and water-soluble polymers and contain 40 to less than 100% (in weight) of protein. The final microsphere product that has been stabilized using a crosslinking agent and / or by its exposure to a source of energy, for example heat, does not swell notably in the presence of water (that is, it is not a hydrogel). In particularly preferred embodiments, the soluble polymer in water it is a hydrocarbon based polymer. So, in certain Preferred embodiments, the microsphere comprises: (1) a protein, preferably albumin; and (2) a polymer soluble in hydrocarbon-based water, preferably hetalmidon, representing the protein at least 40% and less than 100% by weight of the microsphere. Preferably, the polymer based on hydrocarbons represents more than 0% and less than or equal to 30% by weight of the microsphere. In this and other embodiments, the microspheres preferably they further comprise an active agent, preferably a luteinizing hormone release hormone or an analog Of the same. In general, the microspheres of the invention, when are in contact with an active agent solution, are able to incorporate at least 60%, preferably at least 70%, at less than 80% or at least 90%, and with special preference, at least 95% or at least 98% of the active agent. Optionally microspheres containing the active agent are further stabilized contacting the microspheres with the same type of agent crosslinker and using the same types of conditions here described for the initial stabilization of the microspheres.

In accordance with another aspect of the invention, provides a microsphere that also includes an agent complexing According to this aspect, the microsphere includes: (1) a macromolecule such as a protein (for example albumin, such as described above); (2) at least one polymer soluble in water (for example, hetalmidon (hydroxyethyl starch), PEG / PVP); and (3) A complexing agent. As used here, an agent complexing refers to a molecule that is capable of interacting with a therapeutic agent (set out below) to facilitate the loading, retention and / or otherwise delaying agent release therapeutic from the microsphere (see for example Table 3). As with all aspects of the invention, these microspheres they have a smooth surface that includes multiple holes flutes whose diameter is less than 1,000 angstroms, as determined by gas adsorption techniques for the determination of pore size and preferably do not contain oils or detectable organic solvents.

According to one aspect particularly preferred of the invention, a microsphere is provided which It also includes at least two complexing agents. According to this aspect, the microsphere includes: (1) a macromolecule such as a carrier protein (for example albumin, as described previously); (2) at least one water soluble polymer (for example hetalmidon (hydroxyethyl starch), PEG / PVP); (3) a first complexing agent that is an anionic polysaccharide; and (4) a second complexing agent that is a divalent metal cation selected from the group consisting of calcium, magnesium, zinc, strontium, barium, manganese and iron. One realization Particularly preferred of this aspect of the invention is illustrated in Example 17. Calcium and magnesium are cations preferred divalent metallic.

As used here, a complexing agent refers to a molecule that is able to interact with an agent therapeutic (set out below) to facilitate loading, the retention and / or otherwise delay agent release therapeutic from the microsphere (see for example Table 3). As with other aspects of the invention, these microspheres have preferably a smooth surface that includes multiple holes flutes whose diameter is less than 1,000 angstroms, as determined by gas adsorption techniques for determination pore size and preferably do not contain oils or detectable organic solvents.

A preferred method to incorporate the complexing agent (s) consists of combine the complexing agent (s) with a water soluble polymer in aqueous solution, then add the macromolecule and stabilize the microspheres with heat and / or with crosslinking agents.

In general, the complexing agent is an agent ionic complexing agent (that is, the complexing agent is capable of ionic interaction with a therapeutic agent (discussed below)) or a non-ionic complexing agent (i.e., the complexing agent is capable of a non-ionic interaction (for example hydrophobic, mixed ionic / nonionic) with a therapeutic agent or with another agent complexing). Examples of complexing agents are shown in the Table 3. The ionic complexing agents of the invention are further classify anionic complexing agents (i.e. complexing agents that have a negative charge, such as polysaccharides anionics, for example dextran sulfate, galacturonic acids, alginates, mannuronic acid, guluronic acid, hyaluronic acid, Chondroitin, heparin, chitin, chitosan sulfates, glycosaminoglycans, proteoglycans) and complexing agents cationic (i.e. complexing agents that have a charge positive). Preferred complexing agents of the invention are anionic polysaccharides and divalent metal cations selected from the group consisting of calcium and magnesium.

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In certain embodiments, the complexing agent it is an anionic complexing agent that has a structure of formula I:

(I) POLY- [Y -] n X +

where

POLY represents a main chain of the anionic complexing agent that can be linear or branched;

Y - represents an anionic group, for example sulfate, carboxyl, phosphate, nitrate, carbonate and the like, which can be coupled to a or more of the ramifications of the main chain;

X + represents a cationic group, for example it is a counterion of the group anionic;

n is a number integer from 1 to 10,000, preferably from 5 to 100 and, especially from 5 to 1,000 and in particular 5 to 10,000; Y

when n is greater than 1, the n groups Y - may be the same or different.

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In still other embodiments of the invention, the complexing agent is a cationic complexing agent that has The structure of formula II:

(II) POLY- [X +] n Y -

where

POLY represents a main chain of the cationic complexing agent that can be linear or branched;

X + represents a cationic group, for example an amino group, which can attach to one or more of the chain branches principal;

Y - represents an anionic group, which is a counterion of the group cationic;

n is a number integer from 1 to 10,000, preferably from 5 to 100, especially from 5 to 1,000 and in particular 5 to 10,000; Y

when n is greater than 1, the n X + groups may be the same or different.

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In accordance with another aspect of the invention, provides a microsphere that also includes an active agent (see for example Table 4). The microspheres within which can be loaded the active agent can include one or more complexing agents to facilitate loading and / or to modify the release of the active agent from the microsphere. How alternatively, the active agent can be loaded within microspheres described above without an agent complexing, for example, protein and / or soluble polymers in water of the invention can interact with the active agent for facilitate loading and / or modify its release from the microsphere In general, even if the active agent can be loaded into a microsphere of the invention during the preparation of the microsphere, it is preferable to load the active agent in a preformed microsphere of the invention and in particular loading the active agent in a preformed microsphere containing one or more complexing agents to facilitate loading and / or release prolonged agent. Contrary to the microspheres of hydrogel, the microspheres of the invention do not swell significantly in water and, in addition, the microspheres do not require such swelling to provide prolonged release of the therapeutic protein and / or physiologically active agent from the microsphere

As used herein, an active agent refers to an agent that has a diagnostic or therapeutic activity. Accordingly, an active agent may include a detectable label (for example radioactive) that serves to identify the location of the agent released in vivo ; Active agents also include therapeutic agents, which are used to treat a disease or condition. In certain embodiments, the preferred physiologically active agents are proteins or peptide agents. Such proteins or peptide agents can typically be further divided into categories according to the activity of the agent or the type of disease or condition being treated. The categories of physiologically active agents that can be used in the present invention include, but are not limited to, antibiotics, hematopoietic, anti-infective agents, anti-dementia agents, antiviral, anti-tumor, anti-pyretic, analgesic, anti-inflammatory agents, antiulcer, anti-allergic, anti-depressant agents, psychotropic, cardiotonic agents, antiarrhythmic agents, vasodilators, antihypertensive agents, such as hypotensive diuretics, antidiabetic agents, anticoagulants, cholesterol lowering agents, therapeutic agents for osteoporosis, hormones, vaccines, etc ... (see for example Table 4 ).

The physiologically active peptide or protein which is used in accordance with the present invention is a peptide composed of two or more amino acids. Preferably, this peptide it has a molecular weight greater than 200, for example in the range of approximately 200 to 200,000. Molecular weight range Especially preferred is between about 200 and 100,000. More specific examples of physiologically active agents assets, including non-protein agents, that can be used in With regard to the methods and compositions of the invention, provided in the detailed description of the invention.

According to yet another aspect of the invention, a method for forming a microsphere is provided. He method involves: (1) the formation of an aqueous mixture containing a macromolecule, preferably a protein or an acid nucleic, and a water soluble polymer, preferably a polymer based on hydrocarbons, for example hetalmidon; (2) allow the microspheres are formed in the aqueous mixture; (3) stabilization of the microspheres, preferably putting them in contact with a crosslinking agent and / or exposing them to a source of energy, preferably heat, under conditions sufficient to stabilize the microspheres; the macromolecule being present in the mixture aqueous in an amount sufficient to form a microsphere that It contains at least 40% and less than 100% by weight of macromolecule. Although not linked to a particular theory or mechanism, it think that the microspheres are formed during the mixing stage; however, these initially formed microspheres are transitory and require another stage (for example, the inclusion of a crosslinking agent in the mixture and / or the application of heat or another source of energy) to stabilize the microspheres formed temporarily. Typical methods are provided in the Examples to prepare the microspheres.

According to yet another aspect of the invention, a pharmaceutical composition of the material is provided and the method for manufacturing said composition. In certain embodiments, the composition includes a container containing a single dose of microspheres containing an active agent for treat a state that is treatable through liberation prolonged active agent from the microspheres. The number of microspheres in the single dose depends on the amount of agent active present in each microsphere and of the period of time during which prolonged release is desired. Preferably, it select the single dose to achieve prolonged release of the active agent for a period of approximately 1 to approximately 180 days, selecting the single dose of microspheres so that the desired release profile is achieved To treat the condition.

In accordance with another aspect of the invention, provides a syringe containing any of the microspheres described here. For example, the composition can include a syringe that contains a single dose of microspheres containing an active agent to treat a condition that is treatable through prolonged release of active agent from the microspheres. Preferably, a needle to the syringe, the needle having an inside diameter of caliber 14 to 30.

In particular, the microspheres of the invention can be prepared to have a dimension that allows their supply using a syringe without a needle (MediJector, Derata Corporation, Minneapolis, MN 55427), thus eliminating problems of evacuation inherent in needles that should be discarded as hazardous biological waste. So, according to one aspect particularly preferred of the invention, a needleless syringe containing one or more doses of microspheres that contain an active agent to treat a condition. Can be prepare the microspheres so that quality is obtained suitable for supply by other parenteral routes and not parenteral, such as oral, oral, intrathecal, nasal, pulmonary, transdermal, transmucosal and the like.

In accordance with still other embodiments of the invention, microspheres containing acids are provided nucleic Microspheres that contain nucleic acids include: (1) a nucleic acid (for example, a plasmid, a viral vector, oligonucleotides, RNA, antisense and nonsense nucleic acids); (2) a polycationic polymer (for example polylysine); and (3) a water soluble polymer (as described above). Thus, a method for forming microspheres containing nucleic acids. The method implies: (1) combine, in one or more aqueous solutions, a nucleic acid, a polycationic polymer and a water soluble polymer to form an aqueous mixture that can be mono- or multi-phase; and (2) submit the mixture aqueous to a crosslinking agent and / or a source of energy, low sufficient conditions (eg concentration, time of incubation) to stabilize a microsphere. In the Examples typical methods for microsphere formation are provided of nucleic acids.

These and other aspects of the invention are will describe in more detail below. Throughout this description, all technical and scientific terms have the same sense as commonly understood by an average specialist in the matter to which this invention pertains, unless defined else.

Brief description of the figures

Fig. 1: graph showing the percentage cumulative radiolabelled polyethylene glycol (PEG) and serum albumin  Bovine radiolabeled (BSA) released by the microspheres in function of the square root of time in hours. The black square symbol represents PEG and the white square symbol represents BSA.

Fig. 2 graph showing the percentage cumulative radiolabelled bovine serum albumin (BSA) released by microspheres prepared with three different concentrations of polymer as a function of the square root of time in hours. He Gray square symbol represents a total concentration of polymer 50%, the white square symbol represents a concentration 40% polymer total and the black triangle symbol represents a total polymer concentration of 25%.

Fig. 3 graph showing the percentage cumulative radiolabelled polyethylene glycol (PEG) released by microspheres prepared with three different concentrations of polymer as a function of the square root of time in hours. He White triangle symbol represents a total concentration of 50% polymer, the black square symbol represents a 40% total polymer concentration and white square symbol  represents a total polymer concentration of 25%.

Fig. 4 graph showing the cumulative percentage of radiolabelled bovine serum albumin (BSA) released by microspheres prepared with three different concentrations of polymer, at different incubation temperatures, as a function of the square root of time in hours. The white square symbol represents a total polymer concentration of 25% and an incubation at 58 ° C, the black square symbol represents a total polymer concentration of 25% and an incubation at 70 ° C, the white circle symbol represents a total polymer concentration of 40 % and an incubation at 58 ° C, the black circle symbol represents a total polymer concentration of 40% and an incubation at 70 ° C, the white triangle symbol represents a total polymer concentration of 50% and an incubation at 58 ° C, the black triangle symbol represents a total polymer concentration of 50% and an incubation at 70 ° C, the symbol "X" represents a total polymer concentration of 25% and an incubation at 37 ° C and 58 ° C, and the bold symbol " X " represents a total concentration of polymer of 25% and an incubation at 37 ° C and 70 ° C.

Fig. 5 graph showing the percentage cumulative radiolabelled polyethylene glycol (PEG) released by microspheres prepared with three different concentrations of polymer at different incubation temperatures depending on the square root of time in hours. The symbols are the same as those described in Fig. 4.

Fig. 6 graph showing the percentage cumulative radiolabelled polyethylene glycol (PEG) released by microspheres prepared with three different concentrations of polymer at an incubation temperature that includes 58 ° C in function of the square root of time in hours. The symbol white triangle represents a total polymer concentration of 50% and an incubation at 58 ° C, the black square symbol represents a total polymer concentration of 40% and an incubation at 58 ° C, the gray triangle symbol represents a total concentration of 25% polymer and an incubation at 37 ° C and 58 ° C and the symbol white square represents a total polymer concentration of 25% and an incubation at 58 ° C.

Fig. 7 bar graph showing the amount of genetic product expressed by the activity beta-galactosidase in milliunits depending on the microsphere formation for naked DNA, for liposomes cationic containing DNA and for DNA microspheres.

Fig. 8 percentage release chart cumulative leuprolide acetate from the microspheres with the time in days

Fig. 9 percentage release chart cumulative napharelin acetate as a function of time in hours for three concentrations of zinc sulfate used during the Preparation of the microspheres. The circle represents sulfate of 0.01M zinc; the square symbol represents 0.1M zinc sulfate; Y The triangle symbol represents 1M zinc sulfate.

Fig. 10 shows the prolonged release of estradiol in vitro by the microspheres of the invention.

Fig. 11 shows the prolonged release of estradiol in vivo (serum concentrations) by the microspheres of the invention.

Fig. 12 shows the effectiveness of incorporation of diclofenac sodium by adjusting the conditions of incubation.

Fig. 13 shows a representative profile of size distribution for the microspheres of the invention.

Detailed description

The invention provides methods and compositions for prolonged release of therapeutic and / or diagnostic agents in vivo and / or in vitro . In general, the microspheres have a uniform size and shape, the size ranging from about 0.5 microns to about 20 microns, depending on the manufacturing conditions. The characteristics of the microspheres can be altered during the preparation due to the manipulation of the concentration of the water-soluble polymer, the reaction temperature, the pH, the protein concentration, the crosslinking agent and / or the exposure time of the macromolecule. to the crosslinking agent and / or the energy source.

The microspheres serve a wide variety of separations, diagnostic, therapeutic, industrial, commercial, cosmetic and research purposes or for any purpose that requires the incorporation and stabilization of an active molecule, reagent or medication. Thus, the microspheres of the invention are useful in medical and diagnostic applications such as drug delivery, vaccination, genetic therapy and the formation of tumor or tissue imaging in vivo or histopathology. Consequently, the microspheres are suitable for oral or parenteral administration; mucosal administration; ophthalmic, by intravenous, subcutaneous, intraarticular or intramuscular injection; administration by inhalation; and for topical administration.

According to one aspect of the invention, provides a microsphere that has a smooth surface including  multiple corrugated holes. Each corrugated hole has a diameter less than 1,000 angstroms, as determined by gas adsorption techniques for determining the size of pore by, for example, the BET methodology for analysis of data.

The microspheres are prepared by mixing or dissolving macromolecules with a water soluble polymer or with a mixture of water soluble polymers, such as polymers linear or branched from Table 2, at a pH close to the point isoelectric of the macromolecule. The macromolecule and the mixture of polymers are exposed to a crosslinking agent and / or a source of energy, for example heat, under sufficient conditions such as to stabilize the microspheres. The microspheres separate then of the reagents not incorporated by means of methods of separation, for example filtration or centrifugation.

In general, the macromolecule or the combination of macromolecules constitute at least 40% and less than 100% by weight of the final weight of each microsphere. Preferably, the polymer concentration in the microsphere is greater than 0% e less than or equal to 30% by weight. The types of macromolecules that microspheres form include, but are not limited to, proteins, peptides, hydrocarbons, nucleic acids, viruses or mixtures of same.

Each microsphere consists of macromolecules and of polymeric molecules, which intertwine or intermingle in the microsphere and that are generally distributed homogeneous Although without linking to any theory or mechanism In particular, the internal matrix is thought to be soluble in water and, when solubilized, the internal matrix is diffused by the outer surface under appropriate conditions, as explain in more detail later. The microspheres show a narrow size distribution and generally have a shape uniform. The size distribution is also adjustable through the modification of the conditions and reagents used during the preparation process and can be associated with kinetics release, as will be described later. In general, the microspheres have a diameter of less than 10 µm. See the Figure 13 for a representative size distribution of microspheres of the invention. The uniform shape of the microspheres It is substantially spherical, which is why the microparticles They are also referred to here as "microspheres."

The outer surface of each microsphere is permeable to water and dissolved macromolecules and not only allows that aqueous fluids penetrate the microsphere, but allows also that the solubilized macromolecule and the polymer leave The microsphere The microspheres can be made so that release the macromolecule and the polymer inside the microsphere when in an appropriate aqueous medium, such as body fluids or in a physiologically acceptable buffer, in physiological conditions, for a prolonged period of time, thus providing prolonged release of macromolecules. In addition, the microspheres can be manufactured so that they release the macromolecule without initial explosion or rapid release of the macromolecule Prolonged release is defined here as release of macromolecules over a long period of time. The period of time during which the macromolecule continues being released from the microsphere depends on the characteristics of the macromolecule that is being released and the parameters used to form the microspheres, but in all cases it is greater than that of the free aqueous diffusion of the macromolecule. The microspheres containing pharmaceutical compounds can be manufactured so that they release the pharmaceutical compound with the macromolecule and the polymer, as described previously.

As described briefly above and In more detail, the characteristics of the microspheres can be manipulated during preparation by the adjustment of the type of polymer, of the polymer concentration, of the polymer composition, incubation temperature, pH, of the concentration of macromolecules or the period of time during which the macromolecule is exposed to the energy source.

The microspheres can be administered to a being human or animal through oral or parenteral administration, including intravenous, subcutaneous or intramuscular injection; administration by inhalation; intra-articular administration; mucosal administration; ophthalmic administration; Y topical administration Intravenous administration includes the catheterization or angioplasty. The administration can be with therapeutic and diagnostic purposes, as will be explained later.

Microsphere Formation

The microspheres are obtained by mixing the macromolecules in an aqueous mixture with a water soluble polymer or with a mixture of polymers to form the microspheres and optionally, then contacting the microspheres with a crosslinking agent and / or with a source of energy, preferably heat, under sufficient conditions such as to stabilize the microspheres. Preferably, the solution is an aqueous solution To cause the removal of water or dehydration of the macromolecules, or the solution of macromolecules are added to the polymer or the solution of polymers to the solution of macromolecules. The specialists in the matter also refer to this process as "exclusion of volume. "Although not linked to any theory or mechanism In particular, microspheres are thought to form during mixing stage; however, these microspheres initially formed are transient and require another stage (for example, the inclusion of a crosslinking agent in the mixture and / or the application of heat or other energy source) to stabilize the transiently formed microspheres.

The pH of the solution of macromolecule-polymer fits before, after or during mixing of the polymer with the macromolecule at a near pH to the isoelectric point (pI) of the macromolecule, preferably within a range of 3 to 4 pH units of the pI of the macromolecule, especially between 1.5 and 2 pH units of the pI of the macromolecule

The pH adjustment can be carried out by the addition of an acid, of a base, either in the form of a solution or solid, or of a buffer or other salt or pH adjustment solution at macromolecule solution, polymer solution, or to the mixture of macromolecule and polymer according to methods well known for The specialists in the field. Preferably, the polymer is dissolves in a buffer with a pH close to the pI of the macromolecule and then the polymer solution with the adjusted pH is added to the macromolecule, which has been dissolved in an aqueous solution. PH of the final solution should remain close to the pI of the macromolecule

Then the macromolecule solution is exposed and polymer to a crosslinking agent and / or an energy source, by example of heat, radiation or ionization, alone or in combination with sonication, vortex, mixing or agitation, during a period of predetermined time, to stabilize the microspheres. The resulting microspheres are then separated from any unincorporated component present in the solution by means of physical separation methods well known to specialists in the matter and then they can be washed.

The duration of the incubation depends on the respective concentrations of the polymer and the macromolecule and the energy level of the energy source. The stabilization of microspheres can start immediately under exposure to the power source. Preferably, the macromolecule mixture and polymer is heated to a temperature above the temperature Ambient for approximately 5 minutes up to 24 hours. In especially, the polymer and macromolecules are mixed by stirring or oscillation for 30 minutes, at a temperature located approximately between 37ºC and 70ºC.

Macromolecules

The macromolecules that make up the microsphere consist of any molecule that has a tertiary structure and quaternary or that is capable of having a tertiary structure and Quaternary More preferably, the macromolecule is a biomolecule such as a protein, including enzymes and proteins recombinants, a peptide, a hydrocarbon, a polysaccharide, a hydrocarbon- or polysaccharide-protein conjugate, a nucleic acid, a virus, a viral particle, a conjugate of a small molecule (such as a hapten) and a protein, or its mixtures A compound can be incorporated into the microspheres natural or synthetic pharmaceutical, organic or inorganic or a medication by subjecting the medication to a macromolecule, such as a protein, and then by forming of the microspheres from the complex or conjugate macromolecule-medication. The specialists in the matter will understand that a microsphere can form within a compound unable to have a tertiary and quaternary structure, by incorporating or coupling the compound within a carrier molecule that has a tertiary structure and Quaternary The specialists in the field will also understand that the macromolecule can be a part of a molecule, by example a peptide, a single strand segment of a molecule of double stranded nucleic acid, having a tertiary structure and quaternary. It will also be understood that the term "macromolecule" includes a plurality of macromolecules e includes combinations of different macromolecules, such as a combination of a pharmaceutical compound and an affinity molecule  to direct the pharmaceutical compound towards a tissue, organ or tumor that needs treatment. It will also be understood that a affinity molecule can be the receptor part or the part of the ligand of a receptor-ligand interaction. Examples of ligands that interact with other biomolecules include viruses, bacteria, polysaccharides or toxins that act as antigens to generate an immune response when administered to an animal and It causes the production of antibodies.

Suitable compounds or macromolecules include, but not limited to, Betaxolol?, Diclofenac?, doxorubicin, Rifampin ™, leuprolide acetate, hormone luteinizing hormone (LHRH) releasing, (D-Tryp6) -LHRH, acetate nafarelin, insulin, sodium insulin, zinc insulin, protamine, lysozyme, alpha-lactalbumin, growth factor of basic fibroblasts (bFGF), beta-lactoglobulin, trypsin, carbonic anhydrase, ovalbumin, bovine serum albumin (BSA), human serum albumin (HSA), phosphorylase b, alkaline phosphatase, beta-galactosidase, IgG, fibrinogen, poly-L-lysine, IgM, DNA, acetate desmopressin?, hormone release factor growth (GHRF), somatostatin, antide, Factor VIII, G-CSF / GM-CSF, hormone of human growth (hGH), beta interferon, antithrombin III, alpha interferon, alpha interferon 2b.

Incubation conditions are optimized normally to incorporate approximately 100% of the macromolecule in the reaction mixture by adjusting the pH, the temperature, macromolecule concentration or duration of reaction or incubation. In general, less energy is needed to form microspheres with high concentrations of macromolecule.

Preferably, the microspheres composed of nucleic acids are prepared by first mixing the nucleic acid either with a protein, such as bovine serum albumin, or, such as nucleic acids are anions, with the addition of a cation, such as polylysine, which helps a lot to the formation of microspheres.

As mentioned above, a small molecule or compound unable to have a structure tertiary and quaternary, for example a peptide or a compound pharmaceutical, can be formed within a microsphere by incorporation or coupling of the compound within a molecule carrier that has a tertiary and quaternary structure. This It can be achieved in different ways. For example, you can form the microspheres as described herein, by the use of a macromolecule that has a tertiary structure and quaternary, for example a protein, and then the small molecule or compound binds inside and / or on the surface of the microsphere. Alternatively, the small molecule or compound binds to the tertiary and quaternary structure macromolecule using hydrophobic or ionic interactions and then the microspheres from the complex macromolecule-small molecule by means of the method described here. A third way to obtain microspheres from small molecules consists of preparing microspheres using a macromolecule that has a tertiary and quaternary structure of so that the microsphere has a net charge and then add a small molecule or compound that has an opposite net charge to that the small molecule is physically attracted to the microsphere and remain attached to it, but that can be released over time under appropriate conditions. Alternatively, they can use different types of non-covalent interactions, such as hydrophobic or affinity interactions, to allow fixation and subsequent release of small molecules.

When preparing microspheres that contain proteins, before the addition of polymers during formation  a protein stabilizer can be added from the microspheres, such as glycerol, fatty acids, sugars such as sucrose, ions by example of zinc, sodium chloride or any other stabilizer of protein known to those skilled in the art, for minimize protein denaturation.

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Marked Macromolecule

Before being incorporated into a microsphere, the Macromolecule can be marked with a detectable label. The different types and methods of labeling of proteins and molecules of nucleic acids are well known to specialists in the matter. They will understand that a magnetic substance, such as a metal, is included within the definition of the term brand. By example, you can mark the macromolecule with a substance metallic, for example with a metal, so that the microspheres can separate from other substances in solution with the help of a magnetic device

Several other labels are set forth below. specific or groups of markers. For example, the tag can be a radiolabel such as, but not limited to, [32] P, <3> H, <14> C, <35] S, [125] I or [131] I. May a [32] P mark is conjugated to a protein with a reagent of conjugation or can be incorporated into the sequence of a molecule of nucleic acid by nick-translation (cut and change), by final marking or by incorporation of a nucleotide marked. For example, a <^ [3]} H, <[14]} C, <[35]} S mark it can be incorporated into a nucleotide sequence incorporating a precursor marked or by chemical modification, while a mark, [125] I or [131] I is generally incorporated into a nucleotide sequence by chemical modification. The detection of A brand can be carried out by methods such as counting scintillation, gamma ray spectrometry or autoradiography.

The tag can also be a mark of Nuclear Magnetic Resonance (NMR) or Mass, for example, [13] C or <15] N or [19] O. The detection of such marks It can be performed by Mass Spectrometry or NMR. Can be also use stains, chemiluminescent agents, agents bioluminescent and fluorogens to mark the macromolecule. Examples of stains that serve to mark nucleic acids include ethidium, acridine, propidium bromide and other dyes interleaving, and 4 ', 6'-diamidino-2-phenylindole (DAPI) (Sigma Chemical Company, St. Louis, Mo.) or other dyes for nucleic acids. Examples of fluorogens include fluorescein and derivatives, phycoerythrin, allophycocyanin, rhodamine, Texas Red or other fluorogens Fluorogens are generally bound by chemical modification Staining marks can be detected in a spectrophotometer and those of fluorogens in a detector fluorescence.

The macromolecule can also be marked with a chromogen (enzyme substrate) to provide an enzyme or affinity mark, or with an enzyme. As an alternative, the macromolecule can be biotinylated so that it can be used in a biotin-avidin reaction, which can be coupled also to a brand such as an enzyme or a fluorogen. The macromolecule can be labeled with peroxidase, alkaline phosphatase or with other enzymes that produce a chromogenic reaction or Fluorogenic when added to the substrate.

You can also carry out labeling by incorporation of any modified base, amino acid or precursor containing any brand, incorporating a modified base or a amino acid containing a chemical group recognizable by specific antibodies, or by detecting any bound antibody complex, by various means, including immunofluorescence or immunoenzymatic reactions. These brands can be detected by enzyme binding immunoassays (ELISA) or by detecting a color change with the help of a spectrophotometer

Coated Microspheres

To the outer surface of the microspheres other molecules, other than the macromolecules of which are composed of microspheres, according to known methods by specialists in the field, to "cover" or "decorate" the microspheres. The ability of molecules to joining the outer surface of the microsphere is due to high concentration of the macromolecule in the microsphere. These molecules join together with the objective of, for example, facilitating targeting (target location), improve receptor mediation and to escape endocytosis or destruction. For example, Biomolecules such as phospholipids can bind to the surface of the microsphere to prevent endocytosis by endosomes; receptors, antibodies or hormones can bind to the surface to promote or facilitate the targeting of the microsphere towards the desired organ, tissue or cells of the body; and polysaccharides, for example glucans, can be attached to the outer surface of the microsphere to increase or avoid being ingested by macrophages

In addition, to the outer surface or within Microspheres can bind one or more segmentable molecules. The segmentable molecules are designed so that the microspheres are direct first to a predetermined site under conditions appropriate biological and then, upon exposure to a change in biological conditions, for example a change in pH, the molecules they are segmented, causing the release of the microsphere from the target site. In this way, the microspheres are attached or are ingested by cells due to the presence of bound molecules to the surface of the microspheres. When the molecule is segmented, the microspheres remain at the desired site, for example within of the cytoplasm or cell nucleus, and can release the macromolecules of which they are composed. This is particularly useful for the supply of medicines, containing the microspheres a medicine that is directed towards a specific site in need of treatment, being able to medicate slowly release on that site. The segmentation site Preferred is a diester bond.

The microspheres can also be coated with one or more stabilizing substances, which they can be particularly useful for long storage term in parenteral administration or for oral delivery, allowing the passage of the microspheres through the stomach or intestine without dissolving. For example, microspheres intended for oral delivery can be stabilized with a coating of a substance such as mucin, a secretion that contains mucopolysaccharides produced by goblet cells intestinal, submaxillary glands and other glandular cells mucous

In addition, the particles may be coated. non-covalently with compounds such as fatty acids or lipids The coating can be applied to the microspheres by immersion in the coating substance solubilized, by spraying the microspheres with the substance or by other methods well known to specialists in the matter.

In some of the preferred embodiments, the microspheres of the invention include a protein and at least one water soluble polymer. As stated above, the microspheres are formed by contacting the protein and at least a water soluble polymer under aqueous conditions to form the microspheres, and the microspheres are then stabilized by chemical crosslinking or by exposure to an energy source, preferably heat, or both, under conditions (for example, concentration, temperature) resulting in microspheres resistant to physical and chemical treatments, such as solutions Caustic and sonication. The Examples describe the particular conditions to form the microspheres representative of the invention. In the preferred embodiments, The formation reaction is carried out without adding oils or organic solvents

The protein component of the microsphere can be a carrier protein or a therapeutic protein (see for example Table 1) representing from about 40 to less than 100% (% by weight) of the microsphere.

As used herein, a "carrier protein" refers to a protein that has a molecular weight of at least about 1,500 and that exists as a three-dimensional structure. The carrier protein may also be a therapeutic protein, that is, a protein that has a therapeutic activity; however, in general, the term "carrier protein" will be used in this application to refer to a protein that has the main function of providing a three-dimensional structure, for the purpose of microsphere formation, although the carrier protein may also have a secondary function as a therapeutic agent. In certain preferred embodiments, the carrier protein is albumin, in particular human serum albumin. Optionally, the protein microspheres of the invention further include a therapeutic agent, such as a steroid (e.g., estradiol, testosterone, prednisolone, dexamethasone, hydrocortisone, lidocaine base, procaine base) or any other chemical entity known to bind to the albumin, such as GCSF or paclitaxel. In still other embodiments (set forth below), the microspheres of the invention further include a complexing agent (preferably an ionic complexing agent) and, especially a therapeutic agent (preferably a peptide) that is associated with the complexing agent through an interaction. Ionic or non-ionic. In some other embodiments, the protein comprising the matrix is a therapeutic protein (for example a hormone such as insulin or human growth hormone) and the microsphere is constructed and arranged to provide prolonged release of the therapeutic protein in vivo without it being produced. a significant increase in the microsphere volume.

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TABLE 1

one

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In general, the microspheres of the invention will be formed by mixing the protein together with at least one water soluble polymer under suitable conditions, which, preferably, they allow the water soluble polymer to remove the water ("dehydrate") of the protein (see for example the Table 2) within specified or preferred proportions (weight / weight) between the protein and the water soluble polymer (per example, protein ratio range: polymer from about 1: 1 to about 1: 1,000). The proportion Preferred protein: water soluble polymer in the reaction of microsphere formation is in the range of approximately 1: 5 to 1:30. As noted above, a "polymer water soluble "of the invention refers to a polymer or to a mixture of polymers that are preferably capable of interact with the macromolecule (for example, with the protein or with another molecule) to cause volume exclusion. Thus, the preferential process involves the use of a system totally aqueous without involving any oil or organic solvent.

Suitable water soluble polymers include linear or branched soluble polymers, preferably High molecular weight. Polymers can be very soluble in water, moderately soluble in water or slightly soluble in water (soluble in water in a percentage greater than 2% weight / volume). The Preferred water soluble polymers are water soluble or soluble in a water miscible solvent. Soluble polymers in water they can be solubilized first by dissolving them in a water miscible solvent and then combining the solution of polymer with an aqueous solvent. In the realizations particularly preferred, the water soluble polymers of the Invention are selected from those shown in Table 2. In particularly preferred embodiments, the polymer Water soluble is a hydrocarbon based polymer.

Preferred polymers are polyvinylpyrrolidone, polyethylene glycol, dextran, copolymer polyoxyethylene-polyoxypropylene, alcohol polyvinyl, starch, hetalmidon, or mixtures thereof, whose Features are described in more detail below. He polymer or polymer blend can be prepared according to The methods mentioned in US Patent No. 5,525,519 by James E. Woiszwillo, or in the PCT Patent Application No. US93-00073 (International Application No. WO 93/14110), filed on January 7, 1993 and published on January 22, July 1993, by James E. Woiszwillo, joining both here as a reference), where the polymer is dissolved in water or in a aqueous solution, such as a buffer, in a concentration located between 1 and 50 g / 100 ml, depending on the molecular weight of the polymer. The total concentration of preferred polymer in the solution polymer is between 10% and 80%, expressed in weight / volume percentage The preferred concentration of each Polymer in the polymer solution is between 5% and 50%. As stated above, the pH of the solution polymeric can be adjusted before combining with the macromolecule so that the addition of the polymer causes an adjustment of the pH of the solution of the macromolecule, preferably within a unit of pH of pI. The pH can be adjusted during the preparation of the polymer solution, preparing the polymer in a pH buffer predetermined. Alternatively, the pH can be adjusted with an acid. or a base after the preparation of the polymer solution.

The copolymer of polyoxyethylene-polyoxypropylene, also known as poloxamer, it is sold by BASF (Parsippany, N.J.) and is available in a variety of shapes with different percentages Relates of polyoxyethylene and polyoxypropylene within the copolymer

PVP is a non-ionic, hydrophilic polymer, with an average molecular weight ranging from about 10,000 and 700,000 and a chemical formula (C 6 H 9 NO) [n]. The PVP is also know how poly [1- (2-oxo-1-pyrrolidinyl) ethylene],  Povidone?, Polyvidone?, RP 143?, Kollidon?, Peregal ST?, Periston?, Plasdone?, Plasmosan?, Protagent?, Subtosan?, And Vinisil ™. PVP is non-toxic, highly hygroscopic and is Dissolve immediately in water or organic solvents.

Polyethylene Glycol (PEG), also known as poly (oxyethylene) glycol, is a condensation polymer of ethylene oxide and water of general chemical formula HO (CH 2 CH 2 O) [n] H.

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TABLE 2

2

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Dextran is a term applied to polysaccharides produced by bacteria that grow on a sucrose substrate. Native dextrans produced by bacteria such as Leuconostoc mesenteroides and Lactobacteria dextranicum normally have a high molecular weight. Dextrans are normally available and are used in injectable form as plasma expanders in humans.

Polyvinyl alcohol (PVA) is a polymer prepared from polyvinyl acetates by substitution of acetate groups with hydroxyl groups and have the formula (CH 2 CHOH) [n]. Most polyvinyl alcohols are water soluble

Chemical suppliers such as Sigma Chemical Company (St. Louis, Mo.) commercially have PEG, dextran, PVA and PVP.

Preferably, the polymer is a mixture of polymers containing an aqueous solution of a PVP with a weight molecular located between 10,000 and 360.00, in particular 40,000, and a PEG with a molecular weight between 200 and 35,000. Are preferred a PVP with a molecular weight of 40,000 and a PEG with a weight Molecular of 3,500. Preferably, the PVP is dissolved in a acetate buffer and PEG is added to the aqueous PVP solution. The concentration of each polymer is preferably between 1 and 40 g / 100 ml, according to the molecular weight of each polymer. Usually equal concentrations of PVP and PEG provide the mixture of most favorable polymers for microsphere formation.

The preferred alternative polymer is a dextran with a molecular weight between approximately 3,000 and 500,000  dalton.

The volume of polymer added to the macromolecule varies according to size, quantity and concentration of the macromolecule. Preferably, at a volume of solution that contains the macromolecule two volumes of the mixture of polymers with a total concentration of polymers of 5-50% The polymer is present in the liquid phase during the reaction with the macromolecule.

In some of the embodiments and in particular in the embodiments of the microspheres that do not also contain a complexing agent, the water soluble polymer preferably not it is PEG, PVP, dextran, nonylphenol ethoxylates and / or polyvinyl alcohol

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Complexing Agents

In accordance with another aspect of the invention, provides a microsphere that also includes an agent complexing As used herein, a complexing agent will refers to a molecule that is able to facilitate loading, retention and / or otherwise delay agent release therapeutic from the microsphere (see for example Table 3). In Some embodiments, the microsphere according to this aspect includes: (1) a macromolecule such as a protein (for example albumin, as described above); (2) at least one polymer water soluble (eg hetalmidon, PEG / PVP, as has been previously described); and (3) a complexing agent.

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TABLE 3

3

In some embodiments particularly preferred of this aspect of the invention, the microspheres include: (1) a carrier protein; (2) a polymer soluble in Water; (3) a first complexing agent that is a polysaccharide polyanionic such as dextran sulfate, galacturonic acids, alginates, mannuronic acid, guluronic acid, hyaluronic acid, Chondroitin, heparin, chitin, chitosan sulfates, glycosaminoglycans, proteoglycans and complexing agents cationic (i.e. complexing agents that have a charge positive); and (4) a second complexing agent that is a cation divalent metal selected from the group consisting of calcium, magnesium, zinc, strontium, barium, manganese and iron. In the preferred embodiments according to this aspect, the carrier protein is an albumin or an immunoglobulin or other selected protein from among the group consisting of carrier proteins of the Table 1. In general, the microspheres according to this aspect of the invention contain about 40 to less than 100% of protein. In the preferred embodiments according to this aspect, the water soluble polymer is selected from the group consisting in the water soluble polymers of Table 2, preferably a polymer based on a hydrocarbon, in particular a hydroxyethyl starch.

As with other aspects of the invention, preferably these microspheres have a smooth surface that includes multiple fluted holes that are less than 1,000 angstrom, as determined by gas adsorption techniques for the determination of pore size and, preferably, not They contain detectable organic oils or solvents.

A preferred method for incorporating one or more complexing agents consists in combining the one or more complexing agents with a water soluble polymer in solution auosa and with the protein in auosa solution and stabilize the microspheres with heat or with crosslinking agents.

In general, the complexing agent is an agent ionic complexing agent (that is, the complexing agent is capable of ionic interaction) or a non-ionic complexing agent (i.e. complexing agent is capable of a non-ionic interaction (by hydrophobic example)) or an agent that interacts so much ionic as non-ionic (eg IgG). Table 3 shows nonionic complexing agents and complexing agents ionic, such as anionic complexing agents (i.e. complexing agents that have a negative charge) and agents cationic complexing agents (i.e. complexing agents that have a positive charge) typical.

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In certain embodiments, the complexing agent it is an anionic complexing agent that has the structure of formula I:

(I) POLY- [Y -] n X +

in the which

POLY represents a main chain of the anionic complexing agent that can be linear or branched;

Y - represents an anionic group, for example sulfate, carboxyl, phosphate, nitrate, carbonate and the like, which can be coupled to one or more of the ramifications of the main chain;

X + represents a cationic group, for example a counterion of the group anionic;

n is a number integer from 1 to 10,000, preferably from 5 to 100, especially from 5 to 1,000 and in particular 5 to 10,000; Y

when n is greater than 1, the n groups Y - may be the same or different.

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In still other embodiments of the invention, the complexing agent is a cationic complexing agent that has The structure of formula II:

(II) POLY- [X +] n Y -

in the which

POLY represents a main chain of the cationic complexing agent that can be linear or branched;

X + represents a cationic group, for example an amino group, which can attach to one or more of the chain branches principal;

Y - represents an anionic group that is a counterion of the group cationic;

n is a number integer from 1 to 10,000, preferably from 5 to 100, especially from 5 to 1,000 and in particular 5 to 10,000; Y

when n is greater than 1, the n X + groups may be the same or different.

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Active Agents

In accordance with another aspect of the invention, provides a microsphere that also includes an active agent. In Table 4 shows the typical categories of agents assets.

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TABLE 4

4

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The microspheres in which the active agent may include a complexing agent to facilitate loading and / or modifying the release of the active agent from the microsphere Alternatively, the active agent can be loaded into the microspheres described above, which lack a complexing agent, for example, protein and / or polymers Water soluble of the invention can interact with the agent active to facilitate loading and / or modify its release from the microsphere In general, although the active agent can be loaded into a microsphere of the invention during its preparation is it is preferable to load the active agent in a preformed microsphere of the invention and, more preferably, loading the active agent in a preformed microsphere containing one or more complexing agents to facilitate loading and / or prolonged release of the agent. Contrary to the hydrogel microspheres, the microspheres of the invention does not increase significantly in volume in water and, In addition, stabilized microspheres do not need to increase volume in order to provide prolonged release of the therapeutic protein and / or physiologically active agent from the microsphere

As used herein, an active agent refers to an agent that has diagnostic or therapeutic activity. Accordingly, an active agent optionally includes a detectable label (for example, a radioactive label) that serves to identify the location of the agent released in vivo ; Active agents also include therapeutic agents that are used to treat a disease or condition. In certain embodiments, the preferred physiologically active agents are protein or peptide agents. These protein or peptide agents can typically be further divided into categories according to the activity of the agent or the type of disease or condition being treated. The physiologically active agent that can be used in the present invention includes, but is not limited to, the categories of antibiotics, hematopoietic, anti-infective agents, anti-dementia agents, antiviral agents, anti-tumor agents, antipyretic agents, analgesics, anti-inflammatory agents, antiulcer agents, anti-allergic agents, antidepressants, psychotropic agents, cardiotonics, antiarrhythmic agents, vasodilators, antihypertensive agents such as diuretics for hypotensive agents, antidiabetic agents, anticoagulants, cholesterol reducing agents, therapeutic agents for osteoporosis, hormones, vaccines, etc. (see for example Table 4). Although specific examples of active agents (eg, peptide and non-peptide agents) for use in accordance with this invention are mentioned below, this does not mean that other peptide or non-peptide agents are excluded. These active agents can be natural, recombinant or chemically synthesized substances.

The physiologically active agents of the invention include protein or peptide agents, as well as agents non-protein or non-peptide. For easier exposure, these protein or peptide agents are collectively referred to herein peptide agents; non-protein and non-peptide agents are together they will call non-peptide agents here. Agents not Typical peptides include the following non-limiting categories  of agents: a) nucleotides and nucleic acids; b) hydrocarbons and polysaccharides; c) virus and viral particles; d) conjugates or complexes of small molecules and proteins or mixtures thereof; and e) natural or synthetic pharmaceuticals, organic or inorganic Another description of these and other agents that may be used in accordance with the methods and compositions of the The present invention is described in US Pat. Nos. 5,482,706; 5,514,670 and 4,357,259, whose content in its entirety It is incorporated here as a reference.

Physiologically active peptide agents Preferred include peptide hormones, cytokines, factors of growth, factors that act on the cardiovascular system, factors that act on the central nervous systems and peripheral factors that act on humoral electrolytes and organic heme substances, factors that act on the bones and skeleton, factors that act on the gastrointestinal system, factors that act on the immune system, factors that act on the respiratory system, factors that act on the genital organs and enzymes.

Typical hormones include insulin, hormone growth, parathyroid hormone, hormone releasing hormone luteinizing (LH-RH), adrenocorticotropic hormone (ACTH), amylin, oxytocin, luteinizing hormone, (D-Tryp6) -LHRH, acetate nafarelin, leuprolide acetate, follicle stimulating hormone, glucagon, prostaglandins, PGE1, PGE2 and other acting factors on the genital organs and their derivatives, analogs and congeners. As analogs of said LH-RH may be mentioned known substances, for example those described in the U.S. Patent Nos. 4,008,209, 4,086,219, 4,124,577, 4,317,815 and 5,110,904,

Typical antibiotics include tetracycline, aminoglycosides, penicillins, cephalosporins, medications sulfonamide, chloramphenicol sodium succinate, erythromycin, vancomycin, lincomycin, clindamycin, nystatin, Amphotericin B, amantidine, idoxuridine, acid p-aminosalicylic, isoniazid, rifampin, antinomycin D, mitramycin, daunomycin, adriamycin, bleomycin, vinblastine, vincristine, procarbazine, imidazolcarboxamide.

Hematopoietic or thrombopoietic factors Typical include, among others, erythropoietin, stimulating factor of granulocyte colonies (G-CSF), factor macrophage-granulocyte stimulant (GM-CSF) and colony stimulating factor of macrophages (M-CSF), factor preparation leukocyte proliferation (Leucoprol, Morinaga Milk), thrombopoietin, platelet proliferation stimulating factor, megakaryocyte proliferation factor (stimulant) and factor VIII.

Typical antidemence agents include selegelene.

Typical antiviral agents include amantidine and protease inhibitors.

Typical antitumor agents include doxorubicin, daunorubicin, taxol and methotrexate. The Typical antipyretics and analgesics include aspirin, Motrina, Ibuprofin, naprosin, Indocin and acetaminophen.

Typical anti-inflammatory agents include NSAIDS, aspirin, steroids, dexamethasone, hydrocortisone, prednisolone and diclofenac sodium.

Typical antiulcer agents include famotidine, cimetidine, nizatidine, ranitidine and sucralfate.

Typical antiallergic agents include antihistamines, diphenyramine, loratadine and chlorpheniramine.

Antidepressant and psychotropic agents Typical include lithium, amitriptaline, tricyclic antidepressants, Fluoxetine, Prozac and paroxetine.

Examples of cardiotonics include digoxin.

Typical antiarrhythmic agents include metoprolol and procainamide.

Typical vasodilators include Nitroglycerin, nifedipine and Isosorbide dinitrate.

Typical diuretics include hydrochlorothiazide and furosemide.

Typical antihypertensive agents include Captopril, nifedipine and atenolol.

Typical antidiabetic agents include Gucozide, chloropropamide, metformin and insulin.

Typical anticoagulants include warfarin, Heparin and hirudin.

Typical cholesterol lowering agents They include lovastatin, cholestylamine and clofibrate.

Typical therapeutic agents to treat the osteoporosis and other factors that act on the bones and the skeleton include calcium, alendronate, bone GLa peptide, hormone parathyroid and its active fragments (osteostatin, Endocrinology 129, 324, 1991), associated proliferation and bone formation peptide to histones H4 (OGP, The EMBO Journal 11, 1867, 1992) and their muteins, derivatives and analogues thereof.

Enzymes and typical enzyme cofactors include pancrease, L-asparaginase, hyaluronidase, chymotrypsin, trypsin, tPA, streptokinase, urokinase, pancreatin, collagenase, trypsinogen, chemotrypsinogen, plasminogen, streptokinase, adenyl cyclase and superoxide dismutase (SOD).

Typical vaccines include hepatitis B, MMR (measles, mumps and rubella) and polio vaccines.

Typical immunological adjuvants include Freund's adjuvant, muramyl dipeptides, concanavalin A, BCG and levamisole.

Typical cytokines include lymphokines, monokines, hematopoietic factors, etc. Lymphokines and cytokines useful in the practice of the invention include interferons (for example interferon alpha, beta and gamma), interleukins (for example interleukin 2 to 11), etc. The monokines useful in the practice of the invention include interleukin-1, tumor necrosis factors (by example, TNF alpha and beta), malignant leukocyte inhibitor factor (LIF), etc.

Typical growth factors include nerve growth factors (NGF, NGF-2 / NT-3), growth factor epidermal (EGF), fibroblast growth factor (FGF), insulin analog growth factor (IGF), transforming growth (TGF), cell growth factor platelet derived (PDGF), hepatocyte growth factor (HGF), etc.

Typical factors that act on the system cardiovascular include those factors that control pressure blood, atherosclerosis, etc., such as endothelin, endothelin inhibitors, described endothelin antagonists in EP 436189, 457195, 496452 and 528312, JP [Open] No. H-3-94692 / 1991 and 130299/1991, enzyme inhibitors that produce endothelin, vasopressin, renin, angiotensin I, angiotensin II, angiotensin III, angiotensin I inhibitor, receptor antagonist angiotensin II, atrial natriuretic peptide (ANP), peptide antiarrhythmic, etc.

Typical factors that act on central and peripheral nervous systems include opioid peptides (for example encephalin, endorphins), neurotropic factor (NTF), Calcitonin gene associated peptide (CGRP), hormone release of thyroid hormone (HRT), salts and derivatives of HRT [JP [Open] No. 50-121273 / 1975 (Pat. Of States United No. 3,959,247), JP [Open] No. 52-116465 / 1977 (U.S. Pat. No. 4,100,152)], neurotensin, etc.

Typical factors that act on the system Gastrointestinal include secretin and gastrin.

Typical factors that act on humoral electrolytes and organic heme substances include factors that control hemagglutination, cholesterol level in plasma or concentrations of metal ions, such as calcitonin, Apoprotein E and hirudin. Laminin and adhesion molecule Intercellular-I (ICAM I) represent the factors of Typical cell adhesion.

Typical factors that act on the kidney and the urinary tract include substances that regulate function renal, such as the brain-derived natriuretic peptide (BNP), urotensin, etc.

Typical factors that act on sensory organs include the factors that control the sensitivity of different organs, such as substance P.

Typical factors that act on the system immune include the factors that control inflammation and malignant neoplasms and the factors that attack microorganisms infectious, such as chemotactic peptides and bradykinins.

Typical factors that act on the system Respiratory include factors associated with responses asthmatic

Proteins or peptides are also included recombinant or chemically synthesized or natural that can act as antigens, such as cedar pollen and ragweed pollen. These factors are well managed separately, combined with haptens or together with an adjuvant in the formulations of agreement with the present invention.

Training, Stabilization and Characterization of Microspheres

The method to form the microspheres of the invention involves: (1) the combination, in one or more solutions aqueous, of a macromolecule such as a protein, a polymer water soluble (for example, a polymer based on a hydrocarbon) and one or more complexing agents, to form a aqueous mixture (as single or multiple phase system); Y, preferably (2) subjecting the aqueous mixture to a crosslinking agent (for example EDC [1-ethyl-3- (3-dimethylaminopropylcarbodiimide]) and / or to a source of energy, for a sufficient time to form a microsphere, in particular a microsphere that has a smooth surface that includes multiple ribbed holes, of diameter less than 1,000 angstroms, as determined by gas adsorption techniques for determining the size of pore. The examples provide typical methods for Prepare the microspheres.

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Particularly Preferred Embodiments They are microspheres that include:

(one)

a carrier protein;

(2)

a water soluble polymer;

(3)

a first complexing agent that is a polyanionic polysaccharide; Y

(4)

a second complexing agent that is a divalent metal cation, preferably calcium or magnesium. Active agents, such as therapeutic or diagnostic agents can be introduced into these microspheres after their formation. The preferred method for formation of these particularly preferred embodiments of the invention involves: (1) the combination, essentially simultaneous, in a or more aqueous solutions, of the carrier protein, the polymer Water soluble, the first complexing agent and the second agent complexing, to form an aqueous mixture; and (2) let the microspheres form. Optionally, the microspheres can also undergo a crosslinking agent for stabilization. By "essentially simultaneous" means that the second agent complexing (preferably calcium or magnesium) is added approximately 30 minutes after the addition of the others training components. Applicants have discovered that the addition of calcium or magnesium to the aqueous mixture, which is not a essentially simultaneous addition, results in the formation of aggregates and other amorphous forms of particles rather than in microspheres of spherical shape, which are formed when calcium is combined or magnesium essentially simultaneously with others components during the process of formation of microspheres

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The microspheres can be stabilized by incubation of the microspheres formed in the presence of an agent crosslinker and / or an energy source (eg heat), for a predetermined period of time. The agents Typical crosslinkers include dialdehydes, amines, ions multivalent, multifunctional molecules that have affinity for the specific reactive groups of the macromolecule being crosslinking, substituted N-maleimides, halides of bifunctional alkyl, aryl halides, isocyanates, acids aliphatic or aromatic dicarboxylic acids, disulfonic acids aliphatic or aromatic, bifunctional imidoesters and vinyl sulfones. Additional crosslinking agents and their use for stabilize the microspheres are described in U.S. 5,578,709 to J. Woiszwillo, whose entire content is incorporated herein by reference. The preferred energy source is heat. However, the subject matter specialists will understand that other sources of energy include heat, radiation and ionization, separately or in combination with sonication, vortex, mixing or agitation. The formation and / or microsphere stabilization can be carried out immediately, exposing them to the power source, or they can need a long exposure to the power source, depending of the characteristics of the components and the conditions. Preferably, the solution mixture is incubated macromolecule-polymer in a water bath at a temperature greater than or equal to 37 ° C and less than or equal to 90 ° C for 5 minutes to 2 hours approximately. In particular, it is incubated the mixture for 5-30 minutes at a temperature between 50ºC and 90ºC. It should be noted that microspheres can form at lower temperatures if a concentration is used greater than macromolecule. The maximum incubation temperature comes determined by the characteristics of the macromolecule and by the Essential function of the microsphere. For example, for a microsphere in which the macromolecule is a protein, is preferably a temperature below about 70 ° C for conserve protein activity.

The microspheres formed are separated from the unincorporated components of the incubation mixture by methods conventional separation, well known to specialists in the matter. Preferably, the incubation mixture is centrifuge so that the microspheres settle to the bottom of the centrifuge tube and unincorporated components remain in the supernatant, which is then removed by decantation. How alternatively, a suspension containing the microspheres formed it is filtered so that the microspheres are retained in the filter and Unincorporated components pass through it.

The additional purification of the microspheres is carried out by washing in a suitable volume of a solution washing The preferred wash solution is a buffer, in in particular an aqueous nonionic solution or a nonionic solution aqueous containing water soluble polymers. Can be repeated washes as required and microspheres can be separate from the wash solution as described previously.

As mentioned above, the microsphere characteristics can be altered by the handling of incubation conditions. For example, the kinetics of microsphere release can be delayed increasing the reaction temperature or extending the time of reaction during the formation of the microspheres. The kinetics of release is also manipulated by selecting different polymers, different concentrations of polymers or different proportions of the polymers used in the formation of microspheres

The size, shape of the microspheres and the Release kinetics can be controlled by adjusting the microsphere formation conditions. For example, the particle formation conditions can be optimized for so that smaller or larger particles are produced or may increase the total incubation time or the temperature of incubation, resulting in particles that have a kinetics of prolonged release

Nucleic Acid Microspheres

In accordance still with other embodiments of the invention, microspheres are provided in which the Macromolecule is a nucleic acid. The microspheres they contain nucleic acids include: (1) a nucleic acid (for example a plasmid, viral vector, oligonucleotide, RNA, nucleic acids antisense and meaningless); (2) one or more polycationic polymers (for example polylysine); and (3) a water soluble polymer (such as described above). So, according to one aspect associated with the invention, a method for forming microspheres that contain nucleic acids. The method implies: (1) combine, in one or more aqueous solutions, one or more acids nucleic, one or more polycationic polymers and one or more polymers water soluble, to form an aqueous mixture; and (2) submit the aqueous mixture to a crosslinking agent and / or an energy source, for a sufficient time, to form a microsphere. In the examples are provided the typical methods of formation of microspheres of nucleic acids.

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Pharmaceutical Compositions

According to yet another aspect of the invention, a pharmaceutical composition of the materials and the method to produce it. The composition includes a container that contains a single dose of microspheres that contain an active agent to treat a condition that is treatable by prolonged release, from the microspheres, of An active agent. The number of microspheres in the single dose depends on the amount of active agent present in each microsphere and the period of time during which the prolonged release Preferably, the dose is selected unique to achieve prolonged release of active agent over a period of about 1 to about 180 days, with the desired release profile.

In accordance with another aspect of the invention, provides the composition contained in a syringe. The Composition includes a syringe that contains a single dose of microspheres containing an active agent to treat a condition that is treatable by prolonged release, from the microspheres of the active agent; and a needle attached to the syringe, where the needle has an inside diameter of 14 gauge to 30

In particular, microspheres can be prepared preferred of the invention to have a dimension such that allow its supply by means of a syringe without needle (MediJector, Derata Corporation, Minneapolis, MN 55427), eliminating thus the waste problems inherent in needles, which must Dispose of as biologically hazardous waste. Like this according to a particularly preferred aspect of the invention, a needleless syringe containing one or more is provided doses of microspheres containing an active agent to treat one condition. The microspheres can be prepared to have adequate quality for supply by other parenteral routes and non-parenteral, such as oral, oral, intrathecal, nasal, pulmonary, transdermal, transmucosal and the like.

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Utilities

In short, you can think about the compositions of the invention as a combination of several components, such as illustrated in the Tables, under conditions to form the microspheres, which, preferably, have the desirable characteristics of a smooth surface, which are quantifiable in terms of determining whether they have been formed (for example, by visual detection) and in terms of determining the diameter of the grooved holes in the microspheres. So in some of its broader aspects, the compositions of the invention are intended for microspheres having the dimensions necessary in the corrugated holes and comprising a macromolecule (preferably a protein from Table 1) and also a water soluble polymer (preferably a polymer of the Table 2). In yet another aspect, the microspheres comprise a macromolecule (preferably a protein from Table 1), a water soluble polymer (preferably a polymer from the Table 2) and a complexing agent (preferably from Table 3). In the particularly preferred embodiments, the microspheres include: (1) a carrier protein; (2) a polymer soluble in Water; (3) a first complexing agent that is a polysaccharide polyanionic; and (4) a second complexing agent that is a cation divalent metal selected from the group consisting of calcium and magnesium In still other aspects, the invention is intended for any of these aspects in which the microspheres further comprise a therapeutic agent (preferably from Table 4). Thus, in certain embodiments of These aspects combine one or more proteins from Table 1 with one or more water soluble polymers of Table 2 and with one or more active agents in Table 4. In still other embodiments of These aspects combine one or more nucleic acids with one or more more polycations of Table 3, with one or more polymers soluble in water from Table 2 and, optionally, with one or more agents Therapeutics of Table 4. Consequently, the methods of invention provide various microspheres that can be designed for different therapeutic and diagnostic applications through selection of the right combination of agents to achieve the therapeutic or diagnostic objective.

When used in therapy, the microspheres of the invention are administered in amounts therapeutically effective. In general, a quantity Therapeutically effective is that amount of active agent necessary to delay onset, inhibit progression or stop quite the particular condition that is being treated. Generally, the therapeutically effective amount will vary with the age, condition and sex of the subject, as well as with nature and scope of the disease in the subject, all determined by a subject matter specialist The dosage can be adjusted by the doctor or the veterinarian, particularly in case of any complication. A therapeutically effective amount of active agent typically ranges from 0.01 mg / kg to approximately 1,000 mg / kg, preferably from about 0.1 mg / kg to about 200 mg / kg and in particular from about 0.2 mg / kg up to about 20 mg / kg, in one or more daily doses, for one or more days, every week, month, every two or three months, and so on.

The microspheres can be administered alone or in combination with other drug therapies as part of a pharmaceutical composition This pharmaceutical composition can include the microspheres in combination with any support physiological and / or pharmaceutically acceptable standard that is known in the art. The compositions must be sterile and contain a therapeutically effective amount of the microsphere in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically acceptable support", as used herein, means one or more solid fillers or compatible liquids, diluents or encapsulating substances that are appropriate for administration to a human or animal being. He term "support" indicates an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the compositions Pharmaceuticals are also able to bind with molecules of the present invention, and with each other, in a way that does not exist interaction, which would substantially impair effectiveness desired pharmaceutical. "Pharmaceutically acceptable" means also a non-toxic material compatible with such a biological system as a cell, a cell culture, a tissue or an organism. The Support characteristics will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, desiccants, bulking agents, propellants, acidifying agents, coating agents, solubilizers and other materials well known in the art. The appropriate support formulations for oral, subcutaneous, intravenous administrations, intramuscular, etc., can be found at Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

A variety of routes are available. administration. The particular mode selected will depend, for course, of the selected particular drug, of the severity of the condition being treated and the necessary dosage for therapeutic efficacy. In general, the methods of invention can be implemented using any mode of administration that is medically acceptable, which means any way that generates effective levels of the compounds active without causing clinically unacceptable adverse effects. These modes of administration include oral, rectal, topical, nasal, intradermal or parenteral. The term "parenteral" includes the subcutaneous, intravenous route, intramuscular or infusion. Oral administration will be preferred for prophylactic treatment due to patient comfort, as well as for dose programming.

Pharmaceutical compositions can present conveniently in unit dosage form and can be prepared by methods well known in the art Pharmaceutical All methods include the stage of associating the a a support, which constitutes one or more accessory ingredients. In In general, the compositions are prepared by associating uniform and intimately the microspheres with a liquid support, a support finely divided solid or both, and then, if necessary, giving Form the product.

The preparations for administration parenteral include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Other examples of solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive, injectable organic esters such as ethyl oleate. The aqueous supports include aqueous solutions, salts and buffers, such as saline and buffered media, solutions and emulsions or alcoholic / aqueous suspensions. Parenteral vehicles include a solution of sodium chloride, Ringer dextrose, dextrose and sodium chloride, fixed or lactated Ringer oils. The intravenous vehicles include nutrient and fluid loads, loads of electrolytes (such as those based on Ringer dextrose) and Similar. Preservatives and others may also be present additives, for example antimicrobial agents, antioxidants, chelants and inert gases and the like. In general, the microspheres can be administered to the subject (any mammalian receptor) using the same modes of administration as those currently used for microparticle therapy in Humans.

The microspheres are useful for a wide variety of separations, for diagnostic, therapeutic, industrial, commercial, cosmetic and research purposes, as discussed in more detail below. For example, for an in vivo diagnostic purpose, the microspheres may include a macromolecule such as an immunoglobulin or a cell receptor labeled with a detectable label. The administration of the labeled microsphere to a patient creates a diagnostic imaging agent for a proliferative disorder, such as cancer, or a tool for the evaluation of the success of a therapeutic agent in reducing the proliferation of cells or organisms. adverse individuals.

For in vitro diagnosis, microspheres containing a macromolecule, such as an immunoglobulin, a cellular receptor or a probe with specific oligonucleotides of the cell or organism under investigation are combined with a test sample, the microspheres are separated from any component not Sample bound and bound molecules are detected by conventional methods.

The microspheres are useful as agents therapeutic and may allow the use of administration alternatives when they include a medication therapeutic and administered to a patient for a release slow or directed supply of the medication to the site that It requires therapy. The microspheres are also useful as therapeutic or prophylactic agents when they include a macromolecule that is itself a therapeutic agent or prophylactic, for example an enzyme or an immunoglobulin. The Slow release of these therapeutic agents is particularly useful for peptides or therapeutic proteins that have a short half-life and must be given by injection

In addition, the microspheres are useful for purification of molecules from a complex mixture, such as reagent for the detection or quantification of a molecule specific or for the production of molecules such as antibodies For example, microspheres that contain a macromolecule such as an immunoglobulin can be fixed to a chromatography column and used in chromatography of immunoaffinity to separate a ligand from a complex mixture. Alternatively, microspheres that include a macromolecule labeled or a mixture of specific labeled macromolecules of different cells or biomolecules, such as cell receptors, can be used to detect changes in the number of cells or biomolecules in response to a particular test condition by means of techniques such as flow cytometry.

In addition, the microspheres can be used as adjuvants for vaccine production, in which they are injected to an animal to study, for example a mouse or rabbit, the microspheres containing antigens, to activate a response enhanced immune production of antibodies to antigen.

Other additional commercial uses include clinical formulations, such as particle formation  enzymatic for addition to detergents; cosmetics, such as formation of collagen particles that must be suspended in a lotion or cream; dyes; and paintings

In Vitro Diagnostics

In Vitro Assays: The microspheres described herein are useful as solid phase particles in an assay, such as an immunosorbent assay associated with enzymes, dot-blot or Western blot, for the detection of a particular target such as a cell, biomolecule or drug. In a biological sample. The microspheres designed for this use are composed of molecules with specific affinities for the target molecule. For example, the macromolecule is an immunoglobulin, a cell receptor or a probe with oligonucleotides and is attached to a test tube or a microtiter plate.

For the detection or quantification of a target molecule of interest, a sample is combined with a solution which contains the microspheres, the macromolecules on the microspheres are reacted with the target molecule, they separate the microspheres of any unbound component from the sample and the microspheres that contain the bound molecules are detected by conventional methods. Microspheres fluorescently colored are particularly useful in the flow cytometry analysis according to well methods known by specialists in the field.

Histopathology: the microspheres described here they are useful as probes or visual markers of pathologies in a histological sample The macromolecules of the microspheres designed for this use are specific to biomolecules expressed during a particular pathological condition and are marked with a detectable mark. For example, the macromolecule is an immunoglobulin, a cell receptor or a probe with specific oligonucleotides of an abnormal cell, such as a rapidly proliferating cell, or of a pathological organism, by Example, a virus.

For the detection of a pathogenic condition, it combine a histological sample with a solution containing the microspheres, the macromolecules marked on the microspheres are they react with the target molecule of interest and the bound microspheres are detected by detecting the brand of according to methods well known to specialists in the matter.

In Vivo Diagnostic Imaging

The microspheres described herein are useful as imaging agents for the in vivo localization of a particular molecule, a cell type or a pathological condition, similar to that described above with respect to its histopathological use. The macromolecules of the microspheres designed for this use are specific to the molecules expressed by a particular cell or by a pathological organism and are marked with a detectable label. For example, the macromolecule is an immunoglobulin, a cell receptor or a probe with oligonucleotides specific to a tumor cell or a pathological organism such as a virus.

The microspheres are used to detect a pathological condition or to check the success of a therapy, by example chemotherapy or surgery, to ensure that the size of a abnormal tissue tumor has decreased or has been removed completely. For this use, a patient receives administration of a microsphere solution, preferably intravenously, to the marked macromolecules of the microspheres a sufficient amount is given time for them to orient themselves towards organ or region of the affected body, the macromolecule undergoes by reaction with a target molecule expressed by the cell or by the organism under investigation, and the linked microspheres are detected by brand detection by techniques conventional imaging, well known to specialists in the field, for example X-rays.

Drug Supply Systems

The microspheres are useful for therapy or prophylaxis when the macromolecule is a therapeutic agent or a pharmaceutical compound that is supplied to a patient and released slowly from the microspheres over time. These microspheres they are particularly useful for the slow release of drugs with a short biological half-life, for example proteins or peptides. If the pharmaceutical compound cannot be formed in a particle, then it complexes on a support such as albumin, and the complex Pharmaceutical-support compound is formed in a microsphere The microsphere can maintain the slow release of the agent throughout the body or may include an affinity molecule specific for a tissue, or target tumor, and be injected into a patient, for the targeted slow release of the therapeutic agent, such as an antitumor, antiviral, antibacterial agent, antiparasitic or antiarthritic, a cytokine, hormone or insulin, directly to the site that needs therapy. As it has been set forth above, the affinity molecule can be segmentable

The microspheres composed of proteins antigenic or polysaccharide-protein conjugates capable of eliciting an immune response are particularly suitable for use as vaccines.

The microspheres are also useful as vehicles for gene therapy or for the production of "genetic vaccines" when they are composed of acids nuclei, for example of DNA or RNA, that are incorporated into the DNA of the patient or are transfected into a target cell to produce a desired protein For example, the polynucleotides that encode virus core proteins such as influenza or HIV human immunodeficiency can be supplied as microspheres for the expression of an antigenic protein. This is advantageous because new vaccines do not need to be developed so often, since viral core proteins mutate in lesser degree that cell surface antigens currently used in vaccines. The nucleic acid microspheres are supplied to mammalian cells a little or so of the same way naked DNA is supplied. The sequence of Desired nucleic acid is inserted into a vector, such as a plasmid from DNA, with a promoter, such as the SV40 promoter or the promoter of cytomegalovirus, and optionally can include a reporter gene, such as beta-galactosidase. The nucleic acid is preferably combined with a support protein and / or a cation such as polylysine to facilitate particle formation, as described above Then the microspheres are administered directly to the patient or are transfected into cells of mammals that are then administered to the patient who needs the therapy or prophylaxis. The nucleic acid microspheres can include a substance such as chloroquine, which allows the nucleic acids leave the cytoplasmic compartments in the cytoplasm so that it can be transcribed and translated more easily by the cells. In addition, the microspheres can be coated of a substance that increases the efficiency of translation or can be coated with a substance that provides the specific direction of the microspheres towards the cell.

Research Applications

The microspheres are useful as tools for research for the purification of biomolecules from complex mixtures, as a reagent for detection or quantification of a biomolecule, or for the production of biomolecules, by example of antibodies.

For example, the microspheres composed of a macromolecule such as an immunoglobulin are attached to a column chromatography and are used in immunoaffinity chromatography to separate a ligand from a complex mixture. The specialists in the matter they will understand that the microsphere, for use in high pressure liquid chromatography, should be held first to a non-compressible solid phase sphere or pearl so that the column lining keep its rigid structure low Pressure.

Alternatively, microspheres are used that include a marked macromolecule or a mixture of macromolecules specific labeled different cells or cell receptors to detect changes in the number of cells or receptors surface cells in response to a particular condition of test, using techniques such as flow cytometry.

The invention will be more fully understood with reference to the following examples. These examples, without However, they are simply intended to illustrate the embodiments of the invention and should not be construed as limiting the scope Of the same.

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Examples

Example one

Preparation of Microspheres that Contain an Agent Pharmacist

Seroalbumin microspheres were prepared Human (HSA) suitable for use as a vehicle in the medication supply

Preparation of HSA Microspheres of Rifampicin TM

Carbonyl diimidazole (124 mg, Sigma was added Chemical Co., St. Louis, Mo.) to a solution of 50 mg of Rifampicin TM antibiotic (3- [4-methylpiperazinyliminomethyl] rifamycin, Sigma Chemical Co., St. Louis, Mo.) in 2 ml of dimethylformamide (DMF). The resulting mixture was allowed to stand at room temperature for four hours To the mixture was added a mixture of 1 ml of human serum albumin (HSA, 25%, Armor Pharmaceutical Co., Collegeville, Pa.) And 2 ml of deionized water. The mixture was left to room temperature overnight. They were added to the 14 ml mixture of a polymer solution containing 25% PVP (40,000 dalton) and 25% PEG (3,350 dalton) in 0.1M NaOAc, pH 4. The mixture was incubated for 30 minutes at room temperature, for 30 minutes at 37 ° C and for 30 minutes at 58 ° C and then cooled to room temperature. The particles were isolated by centrifugation, washed with deionized water three times and resuspended in 20 ml of water. The percentage of HSA incorporated in the particles it was 74% (subjected to the BCA protein assay TM (Pierce, Rockford, III)). The percentage of Rifampicin TM incorporated into the particles was greater than 6.8%. It was determined that  the average particle size was 68 nm in diameter by means of a Coulter TM cell sorter.

Preparation of HSA Microspheres of Virazole TM

Carbonyl diimidazole (100 mg, Sigma was added Chemical Co., St. Louis, Mo.) to a solution of 36 mg of the drug Registered Virazole TM antiviral (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.) In 0.2 ml dimethylformamide (DMF). He let himself rest the resulting mixture at room temperature for four hours. Be added a mixture of 0.2 ml of human serum albumin to the mixture (HSA) (25%, Armor Pharmaceutical Co., Collegeville, Pa.) And 0.4 ml of deionized water. The mixture was incubated for 30 minutes at room temperature, for 30 minutes at 37 ° C and for 30 minutes at 58 ° C and then cooled to room temperature. The particles were isolated by centrifugation, washed with water deionized three times and resuspended in 20 ml of water. He percentage of HSA incorporated into the particles was 61% (submitted to the BCA TM protein assay (Pierce, Rockford, III)). He percentage of registered Virazole TM incorporated into the particles It was 10%.

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Example 2

Polysaccharide fixation to the outer surface of Protein Microspheres

PRP-AH polysaccharide was coupled to the outer surface of two protein microspheres different.

A dihydrazide derivative of adipic acid (AH) from polyiribosylribitol phosphate (PRP) of type B of Haemophilus influenza (Hib), one of the largest organisms causing bacterial meningitis, called PRP-AH, was prepared by coupling PRP to adipic acid (Sigma Chemical Co., St. Louis, Mo.) in the presence of cyanogen bromide (Sigma Chemical Co., St. Louis, Mo.) (PRP was obtained from the Massachusetts Public Health Biologic Laboratory (Jamaica Plain, Mass .)).

PRP-AH coupling to the Microspheres of Ovalbumin

Ovalbumin microspheres were prepared by adding ovalbumin (1%, Sigma Chemical Co., St. Louis, Mo.) to a polymer solution containing 25% PVP (40,000 dalton) and 25% PEG (3,500 dalton). The mixture was incubated. for 30 minutes at room temperature, 30 minutes at 37 ° C and 30 minutes at 58 ° C, causing the formation of microspheres. The particles were collected by centrifugation. It was determined that the mean particle diameter was about 0.068 µm. Ovalbumin particles (1.5 mg) in 1M MES buffer, pH 5.0 (0.1 ml), were combined with PRP-AH (1.5 mg). Subsequently, 2.79 mg of hydrochloride was added. 1-ethyl-3- (3-dimethylaminopropylcarbodiimide) (EDC, Sigma Chemical Company, St. Louis, Mo.). The reaction at room temperature for three hours. The particles were collected by centrifugation and washed three times with buffer 1M MONTH pH 5.0 (400 µl). It was determined that the production of PRP was 25% by means of the free polysaccharide test with antrone described in METHODS IN IMMUNOLOGY AND IMMUNOCHEMISTRY, Vol. II, Williams, C.A. and Chase, M.W. (eds.), 1968, pp. 288-289, Academics Press, NY. It was determined that the protein content was 55% by protein assay BCA TM (Pierce, Rockford, III). The proportion of PRP with respect to the protein was 0.46. The resulting average particle diameter It was 0.067 µm.

The recovery of the free polysaccharide and the polysaccharide charge in the particles (polysaccharide ratio: ovalbú-
mine) depended on the initial proportion between the ovalbumin particles and the polysaccharide. As the proportion of polysaccharide with respect to the ovalbumin particle increased, the recovery of free polysaccharide decreased and the polysaccharide charge on the particles increased as shown in the Example in Table 1 below.

Example Table one

Polysaccharide Recovery and Loading in Microspheres of Ovalbumin

5

PRP-AH coupling to the Microspheres of Toxoid Tetanus

The toxoid tetanus (27 mg / ml, obtained from Massachusetts Public Health Biologic Laboratory (Jamaica Plain, Mass.)) Was combined with two volumes of a polymer solution that it contained 25% of PVP (40,000 dalton) and 25% of PEG (3,500 Dalton), pH 5.0. The mixture was incubated for 30 minutes at room temperature, 30 minutes at 37 ° C and 30 minutes at 58 ° C, causing the formation of microspheres. The particles were collected by centrifugation. It was determined that the average diameter of particle was about 0.082 µm.

The tetanus toxoid particles (0.825 mg) in 1M MES buffer, pH 5.0 (0.1 ml) were combined with PRP-AH (1.5 mg). Subsequently, 2.79 was added mg of hydrochloride 1-ethyl-3- (3-dimethylaminopropylcarbodiimide)  (EDC, Sigma Chemical Company, St. Louis, Mo.). The reaction at room temperature for three hours. The particles were collected by centrifugation and washed three times with buffer 1M MONTH, pH 5.0 (400 µl). It was determined that the production of PRP it was 16% by means of the free polysaccharide test with antrone described in METHODS IN IMMUNOLOGY AND IMMUNOCHEMISTRY, Vol. II, Williams, C.A. and Chase, M.W. (eds.), 1968, pp. 288-289, Academics Press, NY. It was determined that the protein content was 99% by protein assay BCA TM (Pierce, Rockford, III). The proportion of PRP with respect to The protein was 0.1. The resulting average particle diameter It was 0.080 µm.

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Example 3

Release of Radiolabeled Proteins and Polymers from the Microspheres

The microspheres were prepared using a radiolabeled protein (bovine serum albumin, BSA) and a polymer radiolabeled (PEG). Radioactivity release was measured in time function.

The microspheres were prepared by combination of the radiolabeled protein (10 mg / ml of [14] C-BSA, NEN, Boston, Mass.) With two volumes of a polymer solution containing 25% PVP (40,000 dalton) and 25% of [3] H-PEG (3,500 Dalton, NEN, Boston, Mass.), pH 5.0. The mixture was incubated for 30 minutes at 37 ° C, 30 minutes at 58 ° C and 30 minutes at 70 ° C, causing the formation of microspheres. The particles were collected by centrifugation

The protein and polymer were released slowly from the microspheres by adding 500 ml of a phosphate buffered saline (pH 7.4) and incubation of the mixture at 37 ° C, while stirring gently on a stirrer Nutator TM At different times, the particles were precipitated by centrifugation at 8,000 rpm for 10 minutes, removed the supernatant with a pipette and tested the radioactivity by adding a scintillation fluid liquid and count in a liquid scintillation counter. The particles were then resuspended in 500 ml of a solution phosphate buffered saline (pH 7.4) and replenished at 37 ° C with smooth rotation until the next moment. Release kinetics of the radiolabeled protein and polymer during incubation at 37 ° C is shown in Fig. 1.

The microspheres were prepared and subjected to test as to its release as described previously, however, three concentrations were used other than polymer, and the microspheres were formed by incubation at 58 ° C. In the first preparation, 25% of PEG and 25% PVP. In the second preparation, 20% was used of PEG and 20% of PVP. In the third preparation, a 12.5% PEG and 12.5% PVP. The kinetics is shown in Fig. 2 of radiolabelled protein release. The kinetics of Radiolabelled polymer release is shown in Fig. 3.

The microspheres were prepared and subjected to rehearsal once again as to its release as it has described above, three different concentrations were used polymer and microspheres were formed by incubation at 58 ° C, 70 ° C, both at 37 ° C and 58 ° C and at both 37 ° C and 70 ° C. The Radiolabelled protein release kinetics is shown in the Fig. 4. The kinetics of the radiolabeled polymer is shown in Fig. 5. The release of radiolabelled PEG as a function of concentration of polymer is shown in Fig. 6.

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Example 4

Formation of Microspheres that Contain DNA

The microspheres that contain DNA were prepared and transfected into fibroblast cells. The microspheres were analyzed for the effectiveness of protein transfection and expression.

A 0.025 ml aliquot of a solution of 1 mg / ml of a plasmid DNA (pCMV beta Gal, Promega, Milwaukee,  Wis.) Was complexed with 0.025 ml of a 5.0 mg / ml solution of poly-L-lysine that had a range of average molecular weight between 1 kDa and 40 kDa (Sigma Chemical Co., St. Louis, Mo.).

It was added to the DNA complex of plasmid-poly-L-lysine, while rotating, 0.1 ml of a 25% solution (weight / volume) of polyvinylpyrrolidone (average molecular weight of 40 kDa, Spectrum, Gardena, Calif.) and 25% (weight / volume) of polyethylene glycol (mean molecular weight of 3.35 kDa, Spectrum, Gardena, Calif.) In 0.1M sodium acetate, pH 5.5.

The mixture was incubated at 37 ° C for 30 minutes and then at 70 ° C for 30 minutes. Microspheres were formed that They contained DNA. The mixture was centrifuged at 17,500 x g for 10 minutes, the supernatant was aspirated and washed three times particles with 0.3 ml of 10% glycerol (volume / volume) in water deionized The microspheres were resuspended in 0.050 ml of water deionized The microspheres containing DNA were applied to NIH3T3 fibroblast cells and incubated up to 24 hours to allow the uptake of microspheres by cells. The uptake ended by washing the cells three times with a solution phosphate buffered saline (PBS / Ca [2+] + Mg [2+] -free) (GIBCO-BRL, Gaithersburg, Md.) and the addition of Dulbecco's Essential Minimum Medium (DMEM, GIBCO-BRL, Gaithersburg, Md.).

The uptake and expression of pCMV beta Gal DNA were tested for the effectiveness of transfection  and the amount of beta-galactosidase enzyme expressed. The efficiency of transfection was determined by the cell fixation and color development with the substrate of the enzyme beta-galactosidase X-Gal (5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside,  GIBCO-BRL, Gaithersburg, Md.). The amount of expressed beta-galactosidase enzyme was determined by lysis of transfected cells and measurement of Total enzyme activity with the enzyme substrate beta-galactosidase CPRG (red chlorophenol-beta-D-galactopyranoside, Boehringer Mannheim, Indianapolis, Ind.).

Results : The amount of beta-galactosidase enzyme expressed from lysed cells that were transfected by using: 1) naked DNA (no addition); 2) cationic liposomes plus DNA; or 3) the microsphere containing the DNA, prepared as described above, is shown in Fig. 7.

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Example 5

Formation of Microspheres Containing Acetate Leuprolide

Microspheres were prepared containing the leuprolide acetate peptide and human serum albumin. Acetate leuprolide is a generic analog of the hormone releasing the luteinizing hormone, which is a peptide used essentially in Prostate cancer treatment.

A 0.010 ml aliquot of a 10-100 mg / ml solution of leuprolide acetate in water (LHRH, TAP Pharmaceuticals, Deerfield, III) to 0.168 ml of a 2-10% solution (weight / volume) of sulfate dextran in water (average molecular weight 500 kDa), and mixed gently the solution. The leuprolide / dextran solution is added 0.856 ml of an aliquot of a solution that it contained 25% (weight / volume) of polyethylene glycol with a weight average molecular weight of 3.35 kDa (Spectrum, Gardena, Calif.) and 25% (weight / volume) of polyvinylpyrrolidone with an average molecular weight 40 kDa, in 0.1M aqueous solution of sodium acetate, pH 5.5. Be gently mixed the resulting solution and allowed to stand until for 30 minutes A 0.25 ml aliquot of a solution 20% (weight / volume) of human serum albumin (Sigma Chemical Co., St. Louis, Mo.) in water was then added to the solution. It mixed Gently the final solution and placed in a 70 ° C water bath up to 90 ° C for a period of time between 30 minutes and 3 hours. The microspheres formed.

The microspheres were collected by centrifugation at 17.5 K x g for 10 minutes, washed with 0.5 ml of deionized H2O and were collected again by centrifugation

Sterile particles were prepared using the procedure mentioned above and by filtration Sterile of all solutions before use and conduction of all manipulations in open tube in a culture cabin of laminar flow tissue.

In vitro release of leuprolide acetate was measured by centrifugation of the microspheres and resuspension in a phosphate buffered saline release medium. The release kinetics is shown in Fig. 8.

The microspheres were composed of approximately 10% leuprolide acetate, 50% of human serum albumin, 20% dextran sulfate and 20% polyethylene glycol / polyvinylpyrrolidone.

Similar particles were prepared that included also zinc sulfate or caprylic acid, which delayed the protein and peptide release from the microspheres.

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Example 6

Preparation of Bovine Seroalbumin Microspheres Using Polyethylene Glycol and Poloxamer 407

Seroalbumin microspheres were prepared bovine by means of a mixture of polyethylene glycol polymers and poloxamer 407. 1.25 grams polyethylene glycol (PM) 3,550, Sigma Chemical Co., St. Louis, Mo.) and poloxamer 407 (BASF, Parsippany, N.J.) in 100 ml of a 0.1N sodium acetate stopper, pH 5.5, to develop a 12.5% solution. A solution was dissolved 10 mg / ml bovine serum albumin (BSA, Fraction V, Sigma Chemical Co.) in H2O deionized. An aliquot of 400 ml of the BSA solution was combined with 800 ml of the polymer solution. The mixture was stirred. A clear solution formed. It got hot the solution up to 70 ° C for 30 minutes. Formation was observed of particles by the presence of a white suspension milky

The residual polymer solution was removed by centrifugation at 12,500 rpm for 10 minutes and then by solvent decantation. Two washing steps were performed and centrifugation with 10% ethanol in water to remove the polymer additional residual

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Example 7

Preparation of Bovine Seroalbumin Microspheres Using dextran

Seroalbumin microspheres were prepared bovine by using dextran.

12.5 g of dextran (PM 500,000, Sigma Chemical Co., St. Louis, Mo.) in 100 ml of a buffer 0.1M sodium acetate, pH 5.0, to make a 12.5% solution. Bovine serum albumin (BSA, Sigma Chemical Co.) was dissolved in H2O deionized at a concentration of 10 mg / ml. One was placed 400 ml aliquot of the BSA solution in a tube 1.5 ml microcentrifugation. A BSA solution was added to the 800 ml aliquot of the dextran polymer solution. Be stirred the solution. A clear solution formed. He placed the microcentrifuge tube in a 70 ° C water bath for 30 minutes A milky white suspension was observed indicating the microsphere formation

The residual dextran polymer solution was removed by centrifugation at 12,500 rpm for 10 minutes and then by solvent decantation. Two stages were performed wash and centrifuge with 10% ethanol in water to remove the additional residual polymer. The electron micrographs of exploration revealed the formation of microspheres the size of a submicra, often arranged in a structure similar to a strand.

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Example 8

Preparation of Bovine Seroalbumin Microspheres Using Eudragit Registered TM E100

Seroalbumin microspheres were prepared bovine using the Eudragit® E100 polymer. This Polymer is soluble in an organic solvent miscible with water.

The Eudragit® E100 polymer (Rohm, Malden, Mass.) In a 1: 1 solution of sodium acetate buffer 0.1M (pH 5.0) and ethanol. The final pH of the solution was 6.5. A 400 ml aliquot of a 10 mg / ml solution of Bovine serum albumin (BSA, Sigma Chemical Co. St. Louis, Mo.) se combined with 800 ml of the Eudragit® E100 polymer solution. Be formed a clear solution. The BSA / polymer solution was incubated in a water bath at 70 ° C for 30 minutes. One was observed milky white suspension indicating the formation of microspheres The particles were collected by centrifugation for 10 minutes at 8,000 rpm and liquid decantation.

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Example 9

Preparation of Insulin Microspheres Using Dextran

Insulin microspheres were prepared by the use of the polymer dextran.

A 20% solution (weight / weight) of dextran polymer (PM 500,000, Sigma Chemical Co., St. Louis, Mo.) in a sodium acetate buffer pH 5.0. 1.9 ml of H2O deionized to 20.5 mg insulin (Sigma Chemical Co.). Be they added 100 ml of 0.2M HCl to dissolve the insulin. It was placed in a test tube a 400 ml aliquot of the solution insulin. An 800 ml aliquot of the solution was added Dextran to insulin solution. The mixture was stirred. By adding the Dextran solution the mixture became cloudy. They were heated tubes to a final temperature of 70ºC or 90ºC to form insulin microspheres The microspheres were centrifuged at 10,000 rpm for 5 minutes and the liquid was decanted to remove the residual polymer. The microspheres were washed with 10% ethanol in water

The microspheres were placed in a solution phosphate buffered saline pH 7.4 to determine dissolution characteristics The insulin particles formed at a final temperature of 90 ° C they did not dissolve in the solution phosphate buffered saline, while insulin particles prepared at a final temperature of 70 ° C dissolved in 15 minutes Therefore, the stability of insulin particles can be adjusted by varying the incubation temperature used during particle formation.

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Example 10

Preparation of Microspheres Using Various Polymers

Seroalbumin microspheres were prepared human by using nine different polymers or polymer blends.

Procedure : A protein solution (1-5% human serum albumin, pH 4.5-5.5 or bovine serum albumin) was prepared in a buffer. The following polymer solutions were prepared: polyethylene glycol / polyvinylpyrrolidone (PEG 3 kDa / PEG 40 kDa in a 1: 1 mixture); hetalmidon (500 kDa); Pluronic L-101? polymer; dextran (3-500 kDa); dextran sulfate (3-500 kDa); polyvinylpyrrolidone (10-360 kDa); polyethylene glycol / polyvinylpyrrolidone with inulin (a polysaccharide); polyethylene glycol / polyvinylpyrrolidone with the polymer Pluronic L-101? dextran with the Pluronic L-101? polymer. Approximately one volume of protein solution was mixed with two volumes of polymer solution. The mixture was incubated in a water bath at 70 ° C to 90 ° C for thirty minutes. Then the mixture was placed in an ice bath. Microsphere formation was observed.

The microspheres were centrifuged until it was compacted in a granule, the supernatant was decanted and the granule twice in 10% ethanol in aqueous solution to remove the residual polymer solution. Then they washed three times the microspheres with deionized water. The microspheres were used or were tested immediately or lyophilized for subsequent use

Observations : Microspheres prepared by using polymers having a higher molecular weight or a higher concentration of polymers provide a more viscous medium, which results in a more uniform microsphere size distribution. The inclusion of a surfactant such as the Pluronic L-101? Polymer or of a mixture during the formation of the microspheres affected their size. An increase in protein concentration during the formation of the microspheres caused an increase in the incorporation of protein in the microspheres. In general, an increase in the size of the polymer caused an increase in the incorporation of protein in the microspheres. The use of different polymers affected the release kinetics. For example, bovine serum albumin (BSA) microspheres prepared using dextran released approximately 15% less BSA than microspheres prepared using PEG / PVP. However, the release of polysaccharide by protein-polysaccharide microspheres, such as the release of [3] H-inulin by human / inulin seroalbumin microspheres was faster when dextran was used instead of PEG / PVP .

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Example eleven

Preparation of Protein Microspheres Containing Acetate Napharelin Using Various Polymers

Protein microspheres were prepared which they contained the drug nafarelin acetate.

Napharelin acetate is used for Treatment of endometriosis, precocious puberty and cancer of prostate.

Procedure : Human serum albumin microspheres were prepared as described above in Example 10 using nine different polymers or polymer blends. Napharelin acetate (Rocher Laboratories, Nutley, NJ) was dissolved in deionized water to produce 10 mg / ml of a solution.

To 1 mg of napharelin acetate 10 were added mg of human serum albumin microspheres. The mixture was stirred and It was mixed for 16 hours at 4 ° C. A mixture was added to the mixture. 3M ammonium sulfate solution to a final concentration of 0.5M and stirred and mixed for 15 minutes at room temperature. A 1M zinc sulfate solution was added to a final concentration of 0.01M; 0.1M or 1M and stirred and mixed for 1 hour at room temperature.

The mixtures were centrifuged, the supernatant and the granule was resuspended in three deionized water times to wash the microspheres.

Observations : The addition of zinc sulfate reduced the release rate of napharelin acetate by human serum albumin microspheres. The results are shown in Fig. 9.

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Example 12

Preparation of Doxorubicin / Albumin Microspheres

Seroalbumin microspheres were prepared human containing doxorubicin, medication Chemotherapeutic

1 ml of a 250 mg / ml solution of human serum albumin (Sigma, St. Louis, Mo.) in H2O deionized 0.05 ml of a 5 mg / mL solution of doxorubicin was added (Sigma Chemical Co., St. Louis, Mo.) in H2O deionized, se mixed the combined solution and allowed to stand at temperature Ambience for 30 minutes. To the previous solution, they were added 3.0 ml of a solution of 200 mg / ml dextran sulfate (PM 500,000, Sigma Chemical Co., St. Louis, Mo.) in H2O deionized and the resulting solution was mixed and incubated at 37 ° C for 30 minutes Then the solution was incubated at 70 ° C for 30 minutes, after which 1.0 ml of sodium acetate was added 3M, pH 5.0, and the resulting solution was mixed. Then the solution at 90 ° C for 30 minutes. Microspheres formed. Be the microspheres were washed twice with 5.0 ml of H2O deionized

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Example 13

Incorporation of LHRH in Sulfate Microspheres Dextran / Albumin

The luteinizing hormone releasing hormone it was incorporated into microspheres composed of human serum albumin and dextran sulfate microspheres.

At 0.168 ml of a 10% solution (weight / volume) dextran sulfate (average MW 500,000, Sigma Chemical Co., St. Louis, Mo.) in deionized H2O, 0.25 ml of a 20% solution (weight / volume) of human serum albumin (Sigma Chemical Company, St. Louis, Mo.) in H2O deionized. It mixed gently the solution and 0.856 ml of a solution of 25% polyethylene glycol (weight / volume) (mean PM 3.35 kDa, Spectrum, Gardena, Calif.) And 25% polyvinylpyrrolidone (weight / volume) (average PM 40 kDa, Spectrum, Gardena, Calif.) in one 0.1M aqueous sodium acetate solution, pH 5.5.

The resulting solution was mixed gently and placed in a water bath at a temperature between 70ºC and 90ºC for 30 minutes to 3 hours. Microspheres formed. The microspheres were collected by centrifugation at 17.5K x g for 10 minutes, they were resuspended for washing in 0.5 ml of H2O deionized and collected again by centrifugation The microspheres were resuspended in 0.3 ml of a 0.1M sodium acetate solution, pH 5.5. A part was added 0.01 ml aliquot of a 10 mg / ml solution of a hormone luteinizing hormone releasing agent (LHRH, Sigma Chemical Co., St. Louis, Mo.) in deionized H2O and incubated for 16 hours at room temperature so that the LHRH peptide could bind to the microspheres After the 16-hour binding incubation, it is removed unbound LHRH peptide by two wash cycles with H2O deionized and collected by centrifugation. He LHRH incorporation performance was typically 83 to 90%

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Example 14

Incorporation of estradiol in ProMaxx® Microspheres

Human serum albumin microspheres formed by combining 12.5% by weight of polyethylene glycol and the 12.5% by weight of polyvinylpyrrolidone in sodium acetate buffer 50 mM, pH 5.3, and with 1% (weight / volume) of human serum albumin dissolved in aqueous solution. The solution was placed at 70 ° C for 30 minutes The microspheres formed and precipitated in the solution. The microsphere suspension was washed 3 times in water deionized Then estradiol was loaded into the microspheres of human serum albumin at 3 percentages by weight other than load. Be dissolved estradiol in ethanol and incubated in the presence of albumin microspheres. The affinity of estradiol for albumin through hydrophobic interactions resulted in the ability to load estradiol at various weight percentages of load. Microspheres containing estradiol by 10% by weight They contained 1 mg of estradiol and 8.33 mg of human serum albumin (HSA). Microspheres containing estradiol by 49% by weight contained 8.15 mg of estradiol and 8.33 mg of HSA. Microspheres containing estradiol in 77% by weight contained 27.68 mg of estradiol and 8.33 mg of HSA.

The microspheres were released in a solution phosphate buffered saline (PBS) at pH 7.4.

All samples were subjected to rotation at 20 rpm at 37 ° C to ensure constant mixing. One (1) was added ml of PBS to the microspheres, which were placed in tubes of standard polypropylene centrifugation. In each moment, the tubes were centrifuged, the supernatant removed and placed 1ml of the release medium in a sample vial. Each sample of 1 ml was dried under rapid vacuum and resuspended in 200 µl of ethanol. The estradiol released was measured by HPLC.

Prolonged release of estradiol is shown in Fig. 10.

Prolonged release of estradiol was also demonstrated in vivo . The microspheres were injected into five dogs and the resulting serum concentrations were determined by a radioimmunoassay for estradiol. The results are shown in Figure 11. The three groups composed of five dogs all included a control of estradiol, which resulted in the lowest concentrations and the shortest half-life in vivo . ProMaxx®, which are albumin microspheres loaded with estradiol, basically produced higher concentrations of estradiol in plasma, with a noticeably longer half-life. And when the microspheres with estradiol were chemically stabilized with EDC (ethyl dimethylaminopropyl carbodiimide), increased prolonged plasma levels and an increased half-life of estradiol were observed over a period of 43 days.

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Example fifteen

Incorporation of Diclofenac-Na in Microspheres of ProMaxx®

Bovine serum albumin microspheres formed by combining 12.5% by weight polyethylene glycol and a 12.5% by weight of polyvinylpyrrolidone in sodium acetate buffer 50 mM at pH 5.3 and with bovine serum albumin dissolved in solution watery The solution was placed at 70 ° C for 30 minutes. They formed the microspheres and precipitated in the solution. The suspension of microspheres was washed 3 times in deionized water.

The nonsteroidal anti-inflammatory drug, diclofenac-Na, joined the microspheres of albumin by manipulating the degree of ionization of the diclofenac-Na by altering the pH of the solutions incubation. The effectiveness of the incorporation of Diclofenac-Na could be optimized until incorporation of 65% by adjusting the conditions of incubation at a pH shown in Fig. 12.

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Example 16

Formation of Microspheres Containing Acetate Leuprolide

Leuprolide acetate is an analogue of the luteinizing hormone releasing hormone.

An aqueous solution of albumin is added (by example, human, bovine, mouse or rat serum albumin) to a aqueous solution of complexing anionic agent (dextran sulfate molecular weight (MW) 10 kDa at 500 kDa or heparin or heparan sulfate or polyglutamic or polyaspartic acids) with or without a aqueous solution of divalent cations (calcium or magnesium or zinc) a room temperature and the resulting mixture is stirred for a Time period from 5 minutes to 3 hours. To this mixture is added then an aqueous solution of polymer (polyethylene glycol (PEG) from PM 3.5 to 40 kDa; polyvinylpyrrolidone (PVP) of PM 6 to 40 kDa, or mixtures of both PEG and PVP, or starch or hydroxyethylamidon of PM 500 kDa). The concentration of albumin in the final mixture is 5-30 g / l, the complexing ionic agent of 5-30 g / l, that of divalent cations of 0-30 g / l and the polymer concentration is 100 g / l to 400 g / l. The mixture is stirred for a period of 5 minutes at 6 hours at room temperature. The temperature of the mixture is then it rises to one between 37 ° C and 50 ° C for a period of 5 minutes 3 hours The microspheres are subjected to stabilization by the addition of a chemical stabilizer (EDC or other agent binder) or thermal stabilization by heating to a temperature between 70ºC and 100ºC for a period of 0.5 to 6 hours. The mixture is subjected to the stabilizer for a period. from 10 minutes to 16 hours or for thermal stabilization it maintains the temperature at the final value for 10 minutes at 6 hours. The microspheres are then collected and separated from the unincorporated components of the reaction mixture by filtration or centrifugation, with subsequent washing, or by diafiltration The incorporation of leuprolide acetate at microspheres is carried out by adding a solution aqueous solution of leuprolide to an aqueous suspension of microspheres The microsphere concentrations are of 1-50 g / l, those of leuprolide acetate are 1-100 g / l, in a buffered aqueous medium 0-100 mM at a pH between 2.5 and 10.0. After the incubation of leuprolide acetate with the suspension of microspheres for 5 minutes up to 8 hours at 5-50 ° C, the microspheres containing the peptide are wash by means of the methods described above to remove unbound peptide. In some cases, the microspheres containing the peptide undergo another stabilization by repeated exposure to chemical stabilizers, as has been described above, and then washed to remove non-peptide United. The final microspheres that contain the peptide are then resuspended in water or lyophilized. Its effectiveness is determined  pharmaceutical using a pharmacodynamic model, in rats, testosterone suppression during periods of 7 to 180 days. Microspheres containing leuprolide acetate manufactured from according to the previous method they are injected into rats and the serum leuprolide acetate for 7 to 120 days. Be suppresses testosterone from rat serum in injected rats with microspheres containing leuprolide acetate for 7 Up to 120 days These results show prolonged release of physiologically active leuprolide acetate during that time frame.

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Example 17

Examples of ProMaxx Microspheres containing Calcium

The following examples illustrate the conditions specific to form the preferred microspheres of the invention. It should be understood that concentrations, times and pHs may vary within a reasonable experimental range and continue resulting in microspheres that have the characteristics described later. As described in the detailed description of the invention, it is preferred to add divalent metal (preferably calcium or magnesium) during the formation process and that this metal condition takes place within 30 minutes from the addition of the other components that are used to The formation of microspheres. They are provided below. more details regarding the training procedure of microspheres

At 1 volume of a 25% solution (weight / volume) dextran sulfate (average MW 500 kDa) are added approximately 2 volumes of a 25% solution (weight / volume) of albumin (human) and stir the mixture gently during minus 5 minutes In this mixture they are gently shaken approximately 6 volumes of a 36% solution (weight / volume) of hetalmidon in Aqueous 63 mM Na acetate and to this hetalmidon solution is added water while stirring gently. Gently shake the resulting solution for at least 5 minutes, at which time the sulfate mixture is added slowly dextran-albumin. To this mixture, a suitable volume of a solution of CaCl2, ZnCl2 or 1.5M MgCl2 to make a final mixture containing 25 mM or 100 mM metal cation. This mixture is gently stirred from constant way. The temperature of the reaction mixture is then increases to a final temperature of 60-90 ° C for a period of 50-60 minutes The temperature of the mixture is maintained at 60-90 ° C for 30-120 minutes, moment in which an equal volume of a solution is added 25 mM MES-Na + (acid N-morpholinethanesulfonic acid) at room temperature, pH 6.0 ± 0.5 while stirring. Then the microspheres formed undergo diafiltration against 1 vol of 25 mM MES, pH 6.0 ± 0.5. Then, the microspheres are concentrated and diafiltered against 9 volumes of 25 mM MES, pH 6.0 ± 0.5 a room temperature. Charging and diafiltration are carried out. at a pH of 4.5 ± 0.5 in the case of microspheres containing Zn 2+. The microspheres are concentrated by diafiltration. TO the microsphere suspension containing 17.1 g of albumin (human) a solution of 3.43 g of leuprolide acetate is added in 25 mM MONTH, pH 6.0, at a rate of 0.4 liters per minute. The addition of the leuprolide solution is followed by the addition of the MES solution to the acetate microsphere suspension of leuprolide The suspension is mixed for 15 minutes at temperature environment, at which time the suspension is concentrated by diafiltration To this suspension is added a solution of 17.14 g from EDC ((3,3-ethyldimethylaminopropyl) carbodiimide) in 25 mM MONTH, pH 6.0 ± 0.5, at 0.4 liters per minute. The suspension at room temperature while stirring for 180 minutes, after which it is diafiltered against 10 volumes of distilled water and is concentrated by diafiltration. The content in Leuprolide acetate suspension is determined by adding and dilute a 50% solution (weight / volume) of sucrose in water to the final suspension is approximately 3.75-4.025 mg / ml leuprolide acetate in 5% sucrose (weight / volume). This gross suspension is filled then in a vial in an amount of 2.0 ml per vial and lyophilize

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(1) Physical Characteristics

Charging characteristics: the microspheres that contain Mg 2+ and Ca 2+ bind to the drug at a high yield (> 90%), while the microspheres they contain Zn 2+ bind to the drug at a lower yield than 70%

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(2) In Vitro Release

The microspheres containing Mg 2+ and Ca 2+ had similar rates of leuprolide release that did not vary with the concentration of metal cation present during the manufacture of the microspheres. With 25 mM of Zn 2+, the release rate was similar to that of the microspheres containing Ca 2+ and Mg 2+, but with 100 mM of Zn 2+, the magnitude of the initial "explosion" phase was higher.

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(3) In Vivo Performance

In rats, at an identical dosage of medicament, the microspheres containing Ca 2+ and Mg 2+ suppressed serum testosterone to castration levels for at least 4 weeks. The microspheres that contained Zn 2+ did not suppress testosterone for even three weeks in the rat model.

They are set out in the following points of the Description of the preferred aspects of the invention.

TO.

A microsphere that includes:

(one)

a carrier protein;

(2)

a water soluble polymer;

(3)

a first complexing agent that is a polysaccharide polyanionic; Y

(4)

a second complexing agent that is a cation divalent metal selected from the group consisting of calcium and magnesium

B.

The microsphere of point A, characterized in that the Carrier protein is an albumin or an immunoglobulin.

C.

The microsphere according to any of the points above, characterized in that the protein is selected from the group consisting of the carrier proteins of the Table one.

D.

The microsphere according to any of the points above, characterized in that the microsphere contains 40 to less than 100% protein

AND.

The microsphere according to point A, characterized because the water soluble polymer is a polymer based on a hydrocarbon.

F.

The microsphere according to point A, characterized because the water soluble polymer is a hydroxyethyl starch.

G.

The microsphere according to any of the points above, characterized in that the water soluble polymer is select from the group consisting of soluble polymers in water of Table 2, preferably a polymer based on a hydrocarbon and especially hetalmidon.

H.

The microsphere according to point A, characterized because the polyanionic polysaccharide is selected from the group consisting of dextran sulfate, galacturonic acids, alginates, mannuronic acid, guluronic acid, hyaluronic acid, Chondroitin, heparin, chitin, chitosan sulfates, glycosaminoglycans, proteoglycans and complexing agents cationic (i.e. complexing agents that have a charge positive).

I.

The microsphere according to point A, characterized because the polyanionic polysaccharide is sulfate of dextran

J.

The microsphere according to point A, characterized because the divalent metal cation is calcium.

K.

The microsphere according to point A, characterized because the divalent metal cation is magnesium.

L.

The microsphere according to point A, characterized because the microsphere has a smooth surface that includes multiple corrugated holes, each of said having corrugated holes a diameter that is less than 1,000 angstroms.

M.

The microsphere according to point A, characterized because the microsphere does not contain oils or organic solvents detectable

N.

The microsphere according to point A, which comprises also a therapeutic agent.

OR.

The microsphere according to any of the points above, characterized in that the active agent is selected from among the group consisting of the active agents in Table 4, preferably a luteinizing hormone releasing hormone (LH-RH) or an analog thereof, preferably leuproid.

P.

The microsphere according to any of the points above, characterized in that the macromolecule is albumin, preferably human serum albumin and the water soluble polymer is a polymer based on a hydrocarbon, preferably hetalmidon

Q.

The microsphere according to any of the points above, which further comprises a complexing agent, preferably dextran sulfate, and an active agent, preferably leuprolide acetate.

R.

A syringe containing a single dose of the microspheres according to any of the previous points, characterized in that the syringe preferably also includes a needle that has an inner diameter size of 14 to 30

S.

A method to form a microsphere that understands:

(one)

the formation of an aqueous mixture that contains:

(to)

a carrier protein;

(b)

a water soluble polymer;

(C)

a first complexing agent that is a polysaccharide polyanionic; Y

(d)

a second complexing agent that is a cation divalent metal selected from the group consisting of calcium and magnesium;

(2)

let the microspheres form in the mixture watery; Y

(3)

stabilize the microspheres, preferably contacting the microspheres with a crosslinking agent and / or exposing the microspheres to a source of energy, preferably heat, in sufficient conditions to stabilize the microspheres

T.

The method according to point S, characterized in that the formation of the aqueous mixture is carried out by the essentially simultaneous combination of the carrier protein, the water soluble polymer, the first complexing agent and the second complexing agent

OR.

The method according to point S, which further comprises the step of:

(4)

contact the microsphere with a solution of an active agent, preferably an active agent in Table 4, especially a luteinizing hormone releasing hormone or a analogue thereof, and, in particular, leuprolide, to incorporate the active agent in the microsphere.

V.

The method according to point S, characterized in that the step of contacting the microsphere with the agent solution asset results in an incorporation performance of at least the 60%, preferably at least 70%, at least 80%, at least 90% and in particular at least 95% or at least 98% of active agent.

W.

The method according to points S, T or U, which comprises also the step of:

(5)

stabilize the microspheres, preferably contacting the microspheres with a crosslinking agent under sufficient conditions to stabilize the microsphere

Claims (64)

1. Microsphere comprising:

(one)

a protein or carrier molecule selected from among the group consisting of IgG; IgM; insulin; hGH; lysozyme; alpha-lactoglobulin; growth factor of basic fibroblasts; VEGF; chymotrypsin; trypsin anhydrase carbonic; ovalbumin; phosphorylase b; alkaline phosphatase; beta-galactosidase; fibrinogen; poly-L-lysine; DNA; immunoglobulins (eg, antibodies); casein; collagen; Soy protein; and jelly .;

(2)

a water soluble polymer;

(3)

a first complexing agent that is a polysaccharide polyanionic and

(4)

a second complexing agent that is a cation divalent metal selected from the group consisting of calcium and magnesium

2. Microsphere according to claim 1, characterized in that the protein or carrier molecule is immunoglobulin. 3. Microsphere according to claim 1 or 2, characterized in that the microsphere contains from 40 to less than 100% protein. 4. Microsphere according to claim 1, characterized in that the water soluble polymer is a polymer based on a hydrocarbon. 5. Microsphere according to claim 1, characterized in that the water soluble polymer is a hydroxyethyl starch. 6. Microsphere according to any of the preceding claims, characterized in that the water soluble polymer is selected from the group consisting of the water soluble polymers of Table 2, preferably a hydrocarbon-based polymer and especially hetalmidon. 7. Microsphere according to claim 1, characterized in that the polyanionic polysaccharide is selected from the group consisting of dextran sulfate, galacturonic acids, alginates, manuronic acid, guluronic acid, hyaluronic acid, chondroitin sulfates, heparin, chitin, chitosan, glycosaminoglycans , proteoglycans and cationic complexing agents (ie complexing agents that have a positive charge). 8. Microsphere according to claim 1, characterized in that the polyanionic polysaccharide is dextran sulfate. 9. Microsphere according to claim 1, characterized in that the divalent metal cation is calcium. 10. Microsphere according to claim 1, characterized in that the divalent metal cation is magnesium. 11. Microsphere according to claim 1, characterized in that the microsphere has a smooth surface that includes multiple ribbed holes, each of said ribbed holes having a diameter that is less than 1,000 angstroms. 12. Microsphere according to claim 1, characterized in that the microsphere does not contain detectable organic oils or solvents. 13. Microsphere according to claim 1, which It also comprises a therapeutic agent. 14. Microsphere according to any of the preceding claims, characterized in that the active agent is selected from the group consisting of the active agents in Table 4, preferably a luteinizing hormone releasing hormone (LH-RH) or an analogue thereof , preferably leuprolide. 15. Microsphere according to any of the previous claims, further comprising an agent complexing agent, preferably dextran sulfate, and an agent active, preferably leuprolide acetate. 16. Syringe containing a single dose of the microspheres according to any of the preceding claims, characterized in that the syringe preferably further includes a needle having an inner diameter size of caliber 14 to 30. 17. Method to form a microsphere as defines in any one of claims 1 to 15, that understands:

(one)

the formation of an aqueous mixture that contains:

(to)

a protein or carrier molecule selected from among the group consisting of IgG; IgM; insulin; hGH; lysozyme; alpha-lactoglobulin; growth factor of basic fibroblasts; VEGF; chymotrypsin; trypsin anhydrase carbonic; ovalbumin; phosphorylase b; alkaline phosphatase; beta-galactosidase; fibrinogen; poly-L-lysine; DNA; immunoglobulins (eg, antibodies); casein; collagen; Soy protein; and jelly .;

(b)

a water soluble polymer;

(C)

a first complexing agent that is a polysaccharide polyanionic; Y

(d)

a second complexing agent that is a cation divalent metal selected from the group consisting of calcium and magnesium;

(2)

let the microspheres form in the mixture watery; Y

(3)

stabilize the microspheres, preferably contacting the microspheres with a crosslinking agent and / or exposing the microspheres to a source of energy, preferably heat, in sufficient conditions to stabilize the microspheres

18. Method according to claim 17, characterized in that the formation of the aqueous mixture is carried out by essentially combining simultaneously the protein of the carrier molecule or, the water soluble polymer, the first complexing agent and the second complexing agent . 19. Method according to claim 17, which It also includes the step of:

(4)

contact the microsphere with a solution of an active agent, preferably an active agent in Table 4, especially a luteinizing hormone releasing hormone or a analogue thereof, and, in particular, leuprolide, to incorporate the active agent in the microsphere.

20. Method according to claim 19, characterized in that the step of contacting the microsphere with the active agent solution results in an incorporation yield of at least 60%, preferably at least 70%, at least 80% , at least 90% and in particular at least 95% or at least 98% of the active agent. 21. Method according to claim 17, 18 or 19, which also includes the step of:

(5)

stabilize the microspheres, preferably contacting the microspheres with a crosslinking agent under sufficient conditions to stabilize the microsphere

22. Microsphere comprising:

(one)

a carrier protein;

(2)

a water soluble polymer selected from the group consisting of:

to)

hydrocarbon based polymers selected from between methyl cellulose, carboxymethyl cellulose based polymers, dextran, polydextrose, derivatized chitins, chitosan and derivatives thereof;

b)

acrylates-PEG, polyethyleneimine, polyvinyl acetate and derivatives thereof;

C)

poly (vinyl) alcohol, poly (vinyl) phosphate, acid poly (vinyl) phosphonic and derivatives of themselves;

d)

polyacrylic acids and derivatives of themselves;

and)

polyorganic acids, such as polyimaleic acid, and derivatives thereof;

F)

polyamino acids such as polylysine and polyimino acids as polyiminothyrosine, and derivatives of themselves;

g)

block copolymers and copolymers such as poloxamer 407 or Pluronic L-101 TM polymer, and derivatives of the same;

h)

tert-polymers and derivatives of themselves;

i)

polyethers such as poly (tetramethylene ether glycol) and derivatives thereof;

j)

natural polymers such as zein and pululane and derivatives thereof;

k)

polyimides like poly-n-tris (hydroxymethyl) methylmethacrylate and derivatives thereof;

l)

surfactants such as polyoxyethylene sorbitan and derivatives thereof;

m)

polyesters like poly (ethylene glycol) (n) monomethyl ether mono (succinimidylsuccinate) ester and derivatives of themselves;

n)

cyclic and branched polymers such as cyclodextrins and derivatives thereof; Y

or)

polyaldehydes such as poly (perfluoropropylene oxide-b-perfluoroformaldehyde) and derivatives thereof.

(3)

a first complexing agent that is a polysaccharide polyanionic; Y

(4)

a second complexing agent that is a cation divalent metal selected from the group consisting of calcium and magnesium

23. Microsphere according to claim 22, characterized in that the carrier protein is an albumin or an immunoglobulin. 24. Microsphere according to claim 22 or 23, characterized in that the protein is selected from the group consisting of the carrier proteins of Table 1. 25. Microsphere according to any of claims 22 to 24, characterized in that the microsphere contains from 40 to less than 100% protein. 26. Microsphere according to claim 22, characterized in that the water soluble polymer is a hydrocarbon based polymer. 27. Microsphere according to claim 22, characterized in that the polyanionic polysaccharide is selected from the group consisting of dextran sulfate, galacturonic acids, alginates, manuronic acid, guluronic acid, hyaluronic acid, chondroitin sulfates, heparin, chitin, chitosan, glycosaminoglycans , proteoglycans and cationic complexing agents (ie complexing agents that have a positive charge). 28. Microsphere according to claim 22, characterized in that the polyanionic polysaccharide is dextran sulfate. 29. Microsphere according to claim 22, characterized in that the divalent metal cation is calcium. 30. Microsphere according to claim 22, characterized in that the divalent metal cation is magnesium. 31. Microsphere according to claim 22, characterized in that the microsphere has a smooth surface that includes multiple ribbed holes, each of said ribbed holes having a diameter that is less than 1,000 angstroms. 32. Microsphere according to claim 22, characterized in that the microsphere does not contain detectable organic oils or solvents. 33. Microsphere according to claim 22, which It also comprises a therapeutic agent. 34. Microsphere according to any of claims 22 to 33, characterized in that the active agent is selected from the group consisting of the active agents in Table 4, preferably the luteinizing hormone releasing hormone (LH-RH) or an analogue of the same, preferably leuprolide. 35. Microsphere according to any of claims 22 to 34, characterized in that the macromolecule is albumin, preferably human serum albumin, and the water-soluble polymer is a hydrocarbon-based polymer. 36. Microsphere according to any of the claims 22 to 35, further comprising an agent complexing agent, preferably dextran sulfate, and an agent active, preferably leuprolide acetate. 37. Syringe containing a single dose of the microspheres according to any one of claims 22 to 36, characterized in that the syringe preferably further includes a needle having an inner diameter size of caliber 14 to 30. 38. Method for forming a microsphere as defines in any one of claims 22 to 36, that understands:

(one)

the formation of an aqueous mixture that contains:

(to)

a carrier protein;

(b)

a water soluble polymer selected from the group consisting of:

one)

hydrocarbon based polymers, such as methyl cellulose, carboxymethyl cellulose based polymers, dextran, polydextrose, derivatized chitins, chitosan, and starch (included hetalmidon) and derivatives thereof;

2)

acrylates-PEG, polyethyleneimine, polyvinyl acetate and derivatives thereof;

3)

poly (vinyl) alcohol, poly (vinyl) phosphate, acid poly (vinyl) phosphonic and derivatives of themselves;

4)

polyacrylic acids and derivatives of themselves;

5)

polyorganic acids such as polyimaleic acid and derivatives thereof;

6)

polyamino acids such as polylysine and polyimino acids as polyiminothyrosine, and derivatives of themselves;

7)

block copolymers and copolymers such as poloxamer 407 or Pluronic L-101 TM polymer, and derivatives of the same;

8)

tert-polymers and derivatives of themselves;

9)

polyethers such as poly (tetramethylene ether glycol) and derivatives thereof;

10)

natural polymers such as zein and pululane and derivatives thereof;

eleven)

polyimides like poly-n-tris (hydroxymethyl) methylmethacrylate and derivatives thereof;

12)

surfactants such as polyoxyethylene sorbitan and derivatives thereof;

13)

polyesters like poly (ethylene glycol) (n) monomethyl ether mono (succinimidylsuccinate) ester and derivatives of themselves;

14)

cyclic and branched polymers such as cyclodextrins and derivatives thereof; Y

fifteen)

polyaldehydes such as poly (perfluoropropylene oxide-b-perfluoroformaldehyde) and derivatives thereof.

(C)

a first complexing agent that is a polysaccharide polyanionic; Y

(d)

a second complexing agent that is a cation divalent metal selected from the group consisting of calcium and magnesium;

(2)

let the microspheres form in the mixture watery; Y

(3)

stabilize the microspheres, preferably contacting the microspheres with a crosslinking agent and / or exposing the microspheres to a source of energy, preferably heat, in sufficient conditions to stabilize the microspheres

39. Method according to claim 38, characterized in that the formation of the aqueous mixture is carried out by essentially combining the carrier protein, the water soluble polymer, the first complexing agent and the second complexing agent. 40. Method according to claim 38, which It also includes the step of:

(4)

contact the microsphere with a solution of an active agent, preferably an active agent in Table 4, especially a luteinizing hormone releasing hormone or a analogue thereof, and, in particular, leuprolide, to incorporate the active agent in the microsphere.

41. Method according to claim 40, characterized in that the step of contacting the microsphere with the active agent solution results in an incorporation yield of at least 60%, preferably at least 70%, at least 80% , at least 90% and in particular at least 95% or at least 98% of the active agent. 42. Method according to claim 38, 39 or 40, which also includes the step of:

(5)

stabilize the microspheres, preferably contacting the microspheres with a crosslinking agent under sufficient conditions to stabilize the microsphere

43. Microsphere comprising:

(one)

a carrier protein;

(2)

a water soluble polymer;

(3)

a first complexing agent that is a polysaccharide polyanionic selected from the group consisting of acids galacturonic, alginates, manuronic acid, guluronic acid, acid hyaluronic, chondroitin sulfates, chitin, chitosan, glycosaminoglycans, proteoglycans; Y

(4)

a second complexing agent that is a cation divalent metal selected from the group consisting of calcium and magnesium

44. Microsphere according to claim 43, characterized in that the carrier protein is an albumin or an immunoglobulin. 45. Microsphere according to claim 43 or 44, characterized in that the protein is selected from the group consisting of the carrier proteins of Table 1. 46. Microsphere according to any of claims 43 to 45, characterized in that the microsphere contains from 40 to less than 100% protein. 47. Microsphere according to claim 43, characterized in that the water soluble polymer is a hydrocarbon based polymer. 48. Microsphere according to claim 43, characterized in that the water soluble polymer is a hydroxyethyl starch. 49. Microsphere according to any of claims 43 to 48, characterized in that the water soluble polymer is selected from the group consisting of the water soluble polymers of Table 2, preferably a hydrocarbon-based polymer, and in particular, hetalmidon. 50. Microsphere according to claim 43, characterized in that the polyanionic polysaccharide is selected from the group consisting of galacturonic acids, alginates, manuronic acid, guluronic acid, hyaluronic acid, chondroitin sulfates, chitin, chitosan, glycosaminoglycans, proteoglycans and cationic complexing agents (ie complexing agents that have a positive charge). 51. Microsphere according to claim 43, characterized in that the divalent metal cation is calcium. 52. Microsphere according to claim 43, characterized in that the divalent metal cation is magnesium. 53. Microsphere according to claim 43, characterized in that the microsphere has a smooth surface that includes multiple ribbed holes, each of said ribbed holes having a diameter that is less than 1,000 angstroms. 54. Microsphere according to claim 43, characterized in that the microsphere does not contain detectable organic oils or solvents. 55. Microsphere according to claim 43, which It also comprises a therapeutic agent. 56. Microsphere according to any of claims 43 to 55, characterized in that the active agent is selected from the group consisting of the active agents in Table 4, preferably the luteinizing hormone releasing hormone (LH-RH) or an analogue of the same, preferably leuprolide. 57. Microsphere according to any one of claims 43 to 56, characterized in that the macromolecule is albumin, preferably human serum albumin, and the water soluble polymer is a hydrocarbon-based polymer, preferably hetalmidon. 58. Microsphere according to any of the claims 43 to 57, further comprising a complexing agent  and an active agent, preferably leuprolide acetate. 59. Syringe containing a single dose of the microspheres according to any one of claims 43 to 58, characterized in that the syringe preferably further includes a needle having an inner diameter size of caliber 14 to 30. 60. Method to form a microsphere according to claims 43 to 58, comprising:

(one)

the formation of an aqueous mixture that contains:

(to)

a carrier protein;

(b)

a water soluble polymer;

(C)

a first complexing agent that is a polysaccharide polyanionic selected from the group consisting of acids galacturonic, alginates, manuronic acid, guluronic acid, acid hyaluronic, chondroitin sulfates, chitin, chitosan, glycosaminoglycans, proteoglycans; Y

(d)

a second complexing agent that is a cation divalent metal selected from the group consisting of calcium and magnesium;

(2)

let the microspheres form in the mixture watery; Y

(3)

stabilize the microspheres, preferably contacting the microspheres with a crosslinking agent and / or exposing the microspheres to a source of energy, preferably heat, in sufficient conditions to stabilize the microspheres

61. Method according to claim 60, characterized in that the formation of the aqueous mixture is carried out by essentially combining the carrier protein, the water soluble polymer, the first complexing agent and the second complexing agent. 62. Method according to claim 60, which It also includes the step of:

(4)

contact the microsphere with a solution of an active agent, preferably an active agent in Table 4, especially a luteinizing hormone releasing hormone or a analogue thereof, and, in particular, leuprolide, to incorporate the active agent in the microsphere.

63. Method according to claim 62, characterized in that the step of contacting the microsphere with the active agent solution results in an incorporation yield of at least 60%, preferably at least 70%, at least 80% , at least 90% and in particular at least 95% or at least 98% of the active agent. 64. Method according to claim 60, 61 or 62, which also includes the step of:

(5)

stabilize the microspheres, preferably contacting the microspheres with a crosslinking agent under sufficient conditions to stabilize the microsphere